Anti-T. cruzi antibodies and methods of use

ABSTRACT

The present disclosure is directed to reagents and methods of using the reagents to detect epitopes of Trypanosoma cruzi.

CROSS-REFERENCE TO RELATED APPLICATION(S)

This is a continuation of U.S. patent application Ser. No. 14/686,351,filed on Apr. 14, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/353,678, filed on Jan. 19, 2012, now U.S. Pat.No. 9,073,984, which is a continuation of U.S. patent application Ser.No. 12/342,641, filed on Dec. 23, 2008, which claims priority to U.S.Provisional Patent Application No. 61/017,071, filed on Dec. 27, 2007,the entire contents of all of which are fully incorporated herein byreference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Oct. 6, 2016, isnamed 2016_10_06_8506USC3-SEQ-LIST.txt, and is 34,384 bytes in size.

TECHNICAL FIELD

The present disclosure relates to methods, assays and kits for detectingor quantifying Trypanosoma (Schizotrypanum) cruzi antigens.

BACKGROUND

The parasite Trypanosoma (Schizotrypanum) cruzi causes Chagas' disease(American trypanosomiasis) and is endemic in Central and South America,as well as in Mexico. After a mild acute phase, most infected victimsenter an indeterminate phase that is characterized by a lack ofsymptoms, low parasite count, and low titers of anti-T. cruziantibodies. Approximately 10-30% of persons with chronic T. cruziinfections, develop cardiac or gastrointestinal dysfunction.Chemotherapy can cure a substantial number of congenitally infectedinfants and children, but is largely ineffective in adults who harborchronic infections (Coura, J., and S. de Castro. 2002. A critical reviewon Chagas disease chemotherapy. Mem. Inst. Oswaldo Cruz. 97:3-24).Roughly 25,000 of the estimated 12 million people in endemic countrieswho are chronically infected with T. cruzi die of the illness each year,due to cardiac rhythm disturbances or congestive heart failure(Kirchhoff, L. V. 2006. American trypanosomiasis (Chagas' disease). InTropical Infectious Diseases: Principles, Pathogens and Practice. Vol.R. Guerrant, D. Walker, and P. Weller, editors. Churchill Livingstone,N.Y. 1082-1094).

Chagas was named after the Brazilian physician Carlos Chagas, who firstdescribed it in 1909 (Chagas, C. 1909a. Neue Trypanosomen. VorläufigeMitteilung. Arch. Schiff Tropenhyg. 13:120-122; Redhead, S. A., et al.2006. Pneumocystis and Trypanosoma cruzi: nomenclature andtypifications. J Eukaryot Microbiol. 53:2-11). He discovered that theintestines of Triatomidae harbored a flagellate protozoan, a new speciesof the Trypanosoma genus, and was able to prove experimentally that theparasite could be transmitted to marmoset monkeys that were bitten bythe infected bug. Chagas named the pathogenic parasite that causes thedisease Trypanosoma cruzi (Chagas, 1909a) and later that year asSchizotrypanum cruzi (Chagas, C. 1909b. Nova tripanozomiase humana:Estudos sobre a morfolojia e o ciclo evolutivo do Schizotrypanum cruzin. gen., n. sp., ajente etiolojico de nova entidade morbida do homem.Mem. Inst. Oswaldo Cruz. 1:159-218), both names honoring Oswaldo Cruz, aBrazilian physician and epidemiologist who fought epidemics of yellowfever, smallpox, and bubonic plague at the turn of the 20^(th) century.

Charles Darwin might have suffered from this disease as a result of abite from the “Great Black Bug of the Pampas” he received east of theAndes near Mendoza. Darwin reported the episode in his diaries of theVoyage of the Beagle. Darwin was young and in general good health,though six months previously he had been ill for a month nearValparaiso, but in 1837, almost a year after he returned to England, hebegan to suffer intermittently from a strange group of symptoms,becoming incapacitated for much of the rest of his life.

In endemic areas, T. cruzi is transmitted mainly by blood-suckingtriatomine insects. The disease can also be spread by blood transfusion,intravenous drug use, congenital transmission, by sexual activity, organtransplant or through breast milk (Bittencourt, A. L. 1976. CongenitalChagas disease. Am J Dis Child. 130:97-103; Cheng, K. Y., et al. 2007Immunoblot assay using recombinant antigens as a supplemental test toconfirm the presence of antibodies to Trypanosoma cruzi. Clin VaccineImmunol. 14:355-61; Grant, I. H., et al. 1989. Transfusion-associatedacute Chagas disease acquired in the United States. Ann Intern Med.111:849-51; Hoff, R., et al. 1978. Congenital Chagas's disease in anurban population: investigation of infected twins. Trans R Soc Trop MedHyg. 72:247-50; Kirchhoff, L. V. 1989. Is Trypanosoma cruzi a new threatto our blood supply? Ann Intern Med. 111:773-5; Skolnick, A. 1989. Doesinflux from endemic areas mean more transfusion-associated Chagas'disease? Jama. 262:1433). Currently, there is no vaccine against T.cruzi.

Diagnosis of chronic T. cruzi infection reflects the complexity of theparasite's life cycle. During periods of high fever, diagnosis consistssimply of identifying the parasites in blood, cerebrospinal fluid, fixedtissue or lymph nodes; however, during latency and chronic stages ofinfection, the bug is difficult to detect. In xenodiagnosis, theintestinal contents of insect vectors are examined for T. cruzi severalweeks after these parasites feed on the blood of a suspected patient.However, this procedure is laborious, expensive and lacks sensitivity(Segura, E. 1987. Xenodiagnosis. In Chagas' Disease Vectors. Vol. R. R.Brenner and A. M. Stoka, editors. CRC Press, Boca Raton, Fla. 41-45).

In contrast, serologic assays for antibodies to T. cruzi are well suitedfor rapid and inexpensive diagnosis of the infection. These methodsinclude indirect immunofluorescence, indirect hemagglutination,complement fixation and enzyme immunoassay (Cheng, K. Y., et al. 2007Immunoblot assay using recombinant antigens as a supplemental test toconfirm the presence of antibodies to Trypanosoma cruzi. Clin VaccineImmunol. 14:355-61). A persistent problem with conventional assays hasbeen the occurrence of inconclusive and false-positive results (Almeida,I. C., et al. 1997. A highly sensitive and specific chemiluminescentenzyme-linked immunosorbent assay for diagnosis of active Trypanosomacruzi infection. Transfusion. 37:850-7; Kirchhoff et al., 2006; Leiby,D. A., et al. 2000. Serologic testing for Trypanosoma cruzi: comparisonof radioimmunoprecipitation assay with commercially available indirectimmunofluorescence assay, indirect hemagglutination assay, andenzyme-linked immunosorbent assay kits. J Clin Microbiol. 38:639-42).

No assay has been uniformly accepted as the gold standard serologicdiagnosis of T. cruzi infection (Cheng et al., 2007). Assays that aredesigned to detect T. cruzi DNA have been found to be insensitive(Gomes, M. L., et al. 1999. Chagas' disease diagnosis: comparativeanalysis of parasitologic, molecular, and serologic methods. Am J TropMed Hyg. 60:205-10). A radioimmune precipitation assay (RIPA) thatproduces easily interpreted results was developed nearly two decades agoand has been suggested for use as a confirmatory test in the U.S.(Kirchhoff et al., 1989). Its sensitivity and specificity, however, havenot been systematically validated. Moreover, the complexity of the RIPArender its widespread use outside of research settings difficult (Leibyet al., 2000).

Immunoassays designed to detect anti-T. cruzi antibodies present inpatient samples can provide fast and reliable serological diagnosticmethods. Typically, such diagnostic kits use one or more specificantibodies to act as calibrators, positive controls and/or panelmembers. Often, Chagas high-titer human plasma and/or serum is screenedand spiked into the negative control reagent at specific quantities.Chagas quality control reagents, such as positive controls, are humanplasma or serum samples screened for the presence of antibodies againstspecific epitopes. However, using human serum and plasma samples hasseveral significant disadvantages. These include: (1) increasingregulatory concerns, (2) difficulty in sourcing large volume with hightiter and specificity; (3) lot variability; (4) limitations regardingcharacterization; and (5) cost.

Thus, there remains a need in the art for specific antibodies to act ascalibrators, positive controls and/or panel members. The presentdisclosure optionally overcomes or obviates some of the problems ofcurrent T. cruzi immunoassays (namely, increasing regulatory concerns,difficulty in sourcing large volume with high titer and specificity, lotvariability, limitations regarding characterization, and cost) byproviding novel antibodies, cell lines producing these antibodies, andmethods of making these antibodies.

SUMMARY

An object of the disclosure is to provide antibodies, including,recombinant antibodies and chimeric antibodies, that specifically bindTrypanosoma (Schizotrypanum) cruzi antigens and uses thereof.

In accordance with one aspect of the present disclosure, there isprovided recombinant antibodies, including chimeric antibodies, whichare capable of specifically binding to a diagnostically relevant regionof a T. cruzi protein. The antibodies, including chimeric andrecombinant antibodies, selected from the group consisting of anantibody specific for T. cruzi polypeptides comprised by FP3, Pep2, FP10and FRA.

In one aspect of the disclosure, the antibody is an said antibody isselected from the group consisting of:

(a) an antibody that specifically binds to a diagnostically relevantregion of a T. cruzi polypeptide, wherein the T. cruzi polypeptide isFRA and further wherein said antibody has at last one binding constantselected from the group consisting of: an association rate constant(k_(a)) between about 7.0×10⁵M⁻¹ s⁻¹ to about 7.0×10⁶M⁻¹ s⁻¹, andissociation rate constant (k_(d)) between about 4.0×10⁻³ s⁻¹ to about3.0×10⁻¹ s⁻¹ and an equilibrium dissociation constant (K_(D)) betweenabout 5.7×10⁻¹⁰ M to about 4.3×10⁻⁷M;

(b) an antibody that specifically binds to a diagnostically relevantregion of a T. cruzi polypeptide, wherein the T. cruzi polypeptide isPep2 and further wherein said antibody has at least one binding constantselected from the group consisting of: an association rate constant(k_(a)) between about 1.0×10⁶M⁻¹ s⁻¹ to about 8.0×10⁶M⁻¹ s⁻¹; andissociation rate constant (k_(d)) between about 6.0×10⁻³ s⁻¹ to about4.0×10⁻² s¹ and an equilibrium dissociation constant (K_(D)) betweenabout 7.5×10¹⁰M to about 4.0×10⁻⁸M;

(c) an antibody that specifically binds to a diagnostically relevantregion of a T. cruzi polypeptide, wherein the T. cruzi polypeptide isFP10 and further wherein said antibody has at least one binding constantselected from the group consisting of: (a) an association rate constant(k_(a)) between about 5.0×10⁴M⁻¹ s⁻¹ to about 3.0×10⁵M⁻¹ s⁻¹: (b) andissociation rate constant (k_(d)) between about 1.0×10⁻⁴ s⁻¹ to about8.0×10⁻⁴ s⁻¹; and (c) an equilibrium dissociation constant (K_(D))between about 3.3×10⁻¹⁰ M to about 1.6×10⁻⁸M;

(d) an antibody that specifically binds to a diagnostically relevantregion of a T. cruzi polypeptide, wherein the T. cruzi polypeptide isFP3 and further wherein said antibody has at least one binding constantselected from the group consisting of: an association rate constant(k_(a)) between about 2.0×10⁵M⁻¹ s⁻¹ to about 6.0×10⁶M⁻¹ s⁻¹; andissociation rate constant (k_(d)) between about 2.0×10⁻⁵ s⁻¹ to about8.0×10⁻⁴ s¹; and an equilibrium dissociation constant (K_(D)) betweenabout 3.3×10¹²M to about 4.0×10⁻⁹M; and

(e) any combinations of (a)-(d).

In another aspect of the disclosure, the antibody is a chimeric antibodyexpressed by a cell line, wherein the cell line selected from the groupconsisting of PTA-8136, PTA-8138 and PTA-8140. Optionally, the antibodyis expressed by a cell line selected from the group consisting ofPTA-8137, PTA-8139, PTA-8141, and PTA-8142. The antibodies optionallyare monoclonal antibodies, humanized antibodies, single-chain Fvantibodies, affinity maturated antibodies, single chain antibodies,single domain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fv, and anti-idiotypic antibodies, dual-variable domainimmunoglobulins (DVD-Ig®) or fragments thereof.

In another aspect of the disclosure, there is provided animmunodiagnostic reagent that comprises one or more of these antibodies,including chimeric and recombinant antibodies, which are capable ofspecifically binding a diagnostically relevant region of a T. cruziprotein, wherein the antibodies are selected from the group consistingof FP3, Pep2, FP10 and FRA.

In accordance with another aspect of the disclosure, theimmunodiagnostic reagent comprises an antibody selected from the groupconsisting of:

(a) an antibody that specifically binds to a diagnostically relevantregion of a T. cruzi polypeptide, wherein the T. cruzi polypeptide isFRA and further wherein said antibody has at last one binding constantselected from the group consisting of: an association rate constant(k_(a)) between about 7.0×10⁵M⁻¹ s⁻¹ to about 7.0×10⁶M⁻¹ s⁻¹, andissociation rate constant (k_(d)) between about 4.0×10⁻³ s⁻¹ to about3.0×10⁻¹ s⁻¹ and an equilibrium dissociation constant (K_(D)) betweenabout 5.7×10¹⁰M to about 4.3×10⁻⁷M;

(b) an antibody that specifically binds to a diagnostically relevantregion of a T. cruzi polypeptide, wherein the T. cruzi polypeptide isPep2 and further wherein said antibody has at least one binding constantselected from the group consisting of: an association rate constant(k_(a)) between about 1.0×10⁶M⁻¹ s⁻¹ to about 8.0×10⁶M⁻¹ s⁻¹; andissociation rate constant (k_(d)) between about 6.0×10⁻³ s⁻¹ to about4.0×10⁻² s¹ and an equilibrium dissociation constant (K_(D)) betweenabout 7.5×10¹⁰M to about 4.0×10⁻⁸M;

(c) an antibody that specifically binds to a diagnostically relevantregion of a T. cruzi polypeptide, wherein the T. cruzi polypeptide isFP10 and further wherein said antibody has at least one binding constantselected from the group consisting of: (a) an association rate constant(k_(a)) between about 5.0×10⁻⁴M⁻¹ s⁻¹ to about 3.0×10⁵M⁻¹ s⁻¹: (b) andissociation rate constant (k_(d)) between about 1.0×10⁻⁴ s⁻¹ to about8.0×10⁻⁴ s⁻¹; and (c) an equilibrium dissociation constant (K_(D))between about 3.3×10⁻¹⁰ M to about 1.6×10⁻⁸M;

(d) an antibody that specifically binds to a diagnostically relevantregion of a T. cruzi polypeptide, wherein the T. cruzi polypeptide isFP3 and further wherein said antibody has at least one binding constantselected from the group consisting of: an association rate constant(k_(a)) between about 2.0×10⁵ M⁻¹ s⁻¹ to about 6.0×10⁶M⁻¹ s⁻¹ andissociation rate constant (k_(d)) between about 2.0×10⁻⁵ s⁻¹ to about8.0×10⁻⁴ s⁻¹; and an equilibrium dissociation constant (K_(D)) betweenabout 3.3×10⁻¹²M to about 4.0×10⁻⁹M; and

(e) any combinations of (a)-(d).

In accordance with another aspect of the disclosure, theimmunodiagnostic reagent is selected from the group consisting of adetection reagent, a standardization reagent, and a positive controlreagent.

In accordance with another aspect of the disclosure, there is providedantibodies, including chimeric and recombinant antibodies, which arecapable of specifically binding to a diagnostically relevant region of aT. cruzi protein, the region comprising an epitope comprised by an aminoacid sequence selected from the group consisting of an amino acidsequence having at least 80%, at least 90% and at least 95% sequenceidentity with an amino acid sequence as set forth in SEQ ID NO.:2, SEQID NO.:4, SEQ ID NO.:6 and SEQ ID NO.:8. In accordance with anotheraspect of the disclosure, the immunodiagnostic reagent that specificallybinds to a diagnostically relevant region of a T. cruzi protein thatcomprises a chimeric antibody, wherein the chimeric antibodyspecifically binds to an epitope comprised by an amino acid sequenceselected from the group consisting of an amino acid sequencesubstantially identical with an amino acid sequence as set forth in SEQID NO.:2, SEQ ID NO.:4, SEQ ID NO.:6 and SEQ ID NO.:8. The antibodiesoptionally are monoclonal antibodies, humanized antibodies, single-chainFv antibodies, affinity maturated antibodies, single chain antibodies,single domain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fv, and anti-idiotypic antibodies, or fragmentsthereof. In accordance with another aspect of the disclosure, there isprovided an immunodiagnositic reagent that comprises these antibodies.

In accordance with another aspect of the disclosure, there is providedantibodies, including chimeric and recombinant antibodies, andimmunodiagnostic reagents comprising the antibodies, wherein theantibodies comprise a V_(H) region selected from the group consisting ofSEQ ID NO.:10, SEQ ID NO.:14, SEQ ID NO.:18 and SEQ ID NO.:28.

In accordance with another aspect of the disclosure, there is providedantibodies, including chimeric and recombinant antibodies, andimmunodiagnostic reagents comprising the antibodies, wherein theantibodies comprise a V_(L) region selected from the group consisting ofSEQ ID NO.:12, SEQ ID NO.:16, SEQ ID NO.:20 and SEQ ID NO.:26.

In accordance with another aspect of the disclosure, there is providedantibodies, including chimeric and recombinant antibodies, andimmunodiagnostic reagents comprising the antibodies, wherein theantibodies are selected from the group consisting of an antibody thatcomprises a V_(H) region substantially identical to the sequence as setforth in SEQ ID NO.:10 and a V_(L) region comprising an amino acidsequence substantially identical to the sequence as set forth in SEQ IDNO.:12; a V_(H) region substantially identical to the sequence as setforth in SEQ ID NO.:14 and a V_(L) region comprising an amino acidsequence substantially identical to the sequence as set forth in SEQ IDNO.:16; a V_(H) region substantially identical to the sequence as setforth in SEQ ID NO.:18 and a V_(L) region comprising an amino acidsequence substantially identical to the sequence as set forth in SEQ IDNO.:20; a V_(H) region substantially identical to the sequence as setforth in SEQ ID NO.:28 and a V_(L) region comprising an amino acidsequence substantially identical to the sequence as set forth in SEQ IDNO.:26. The antibodies optionally are monoclonal antibodies, humanizedantibodies, single-chain Fv antibodies, affinity maturated antibodies,single chain antibodies, single domain antibodies, Fab fragments, F(ab′)fragments, disulfide-linked Fv, and anti-idiotypic antibodies, orfragments thereof

In accordance with another aspect of the disclosure, there is provided acell line capable of expressing a chimeric antibody that specificallybinds to a diagnostically relevant region of a T. cruzi protein, whereinthe cell line optionally is selected from the group consisting ofPTA-8136, PTA-8138 and PTA-8140. There is also provided a cell line thatis capable of expressing an antibody that specifically binds to adiagnostically relevant region of a T. cruzi protein, wherein the cellline optionally is selected from the group consisting of PTA-8137,PTA-8139, PTA-8141 and PTA-8142.

In accordance with another aspect of the present disclosure, there isprovided a method of standardizing a T. cruzi detection assay comprisingusing as a sensitivity panel an immunodiagnostic reagent optionallycomprising one or more antibodies, including chimeric and recombinantantibodies, that are capable of specifically binding a diagnosticallyrelevant region of a T. cruzi protein. In such a panel, optionally theone or more antibodies are selected from the group consisting of anantibody specific for FP3, Pep2, FP10 and FRA.

In accordance with another aspect of the present disclosure, there isprovided a method for detecting the presence of T. cruzi antigenscomprising contacting a test sample, such as a sample suspected ofcontaining T. cruzi antigens, with an immunodiagnostic reagentcomprising one or more antibodies, including chimeric and recombinantantibodies, which are capable of specifically binding a T. cruziantigen. Optionally the contacting is done under conditions that allowformation of antibody:antigen complexes. Further optionally, the methodcomprises detecting any antibody:antigen complexes formed. Theantibodies optionally are monoclonal antibodies, humanized antibodies,single-chain Fv antibodies, affinity maturated antibodies, single chainantibodies, single domain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fv, and anti-idiotypic antibodies, or fragments thereof

In accordance with another aspect of the present disclosure, there isprovided a method for detecting the presence of T. cruzi antibodiescomprising contacting a test sample, such as a sample suspected ofcontaining antibodies to T. cruzi, with one or more antigens specificfor the T. cruzi antibodies. Optionally this contacting is done underconditions that allow formation of antigen:antibody complexes, andfurther optionally the method comprises detecting the antigen:antibodycomplexes. Still further, the method optionally comprises using animmunodiagnostic reagent comprising one or more antibodies, includingchimeric and recombinant antibodies, wherein each of the antibodies arecapable of specifically binding one of the antigens used in the method,e.g., either as a positive control or standardization reagent.

In accordance with another aspect of the present disclosure, there isprovided a diagnostic kit for the detection of T. cruzi comprising animmunodiagnostic reagent comprising one or more antibodies, includingrecombinant and recombinant chimeric antibodies, which are capable ofspecifically binding a diagnostically relevant region of a T. cruziprotein. In such a kit, the one or more antibodies optionally areselected from the group consisting of an antibody, including chimericand recombinant antibodies, specific for FP3, Pep2, FP10 and FRA. Theantibodies optionally are monoclonal antibodies, humanized antibodies,single-chain Fv antibodies, affinity maturated antibodies, single chainantibodies, single domain antibodies, Fab fragments, F(ab′) fragments,disulfide-linked Fv, and anti-idiotypic antibodies, or fragments thereof

In accordance with yet another aspect of the present disclosure, thereis provided isolated polypeptides that comprise a portion of a chimericantibody that specifically binds to a diagnostically relevant region ofa T. cruzi polypeptide selected from the group consisting of T. cruzipolypeptides comprised by FP3, Pep2, FP10 or FRA polypeptides. Thechimeric antibody optionally is selected form the group consisting of achimeric antibody that specifically binds an epitope comprised by anamino acid sequence selected from the group consisting of an amino acidsequence substantially identical with an amino acid sequence as setforth in SEQ ID NO.:2, SEQ ID NO.:4, SEQ ID NO.:6 and SEQ ID NO.:8. Theisolated polypeptides optionally comprise a V_(H) region selected fromthe group consisting of an amino acid sequence substantially identicalto the sequence as set forth in SEQ ID NO.:10, SEQ ID NO.:14 SEQ IDNO.:18, and SEQ ID NO.:28. The isolated polypeptides optionally comprisea V_(L) region selected from the group consisting of an amino acidsequence substantially identical to the sequence as set forth in SEQ IDNO.:12, SEQ ID NO.:16, SEQ ID NO.:20 and SEQ ID NO.:26. Further, theisolated polypeptides comprise both a V_(H) and V_(L) region selectedfrom the group consisting of a V_(H) region of SEQ ID NO.:10 and a V_(L)region of SEQ ID NO.:12; V_(H) region of SEQ ID NO.:14 and a V_(L)region of SEQ ID NO.:16; V_(H) region of SEQ ID NO.:18 and a V_(L)region of SEQ ID NO.:20; and V_(H) region of SEQ ID NO.:28 and a V_(L)region of SEQ ID NO.:26.

In accordance with another aspect of the disclosure, there is providedisolated polynucleotides that encode a portion of a chimeric antibodythat specifically binds to a diagnostically relevant region of a T.cruzi polypeptide, the T. cruzi polypeptide selected from the groupconsisting of T. cruzi polypeptides comprised by FP3, Pep2, FP10 and FRApolypeptides. The chimeric antibody optionally is selected form thegroup consisting of a chimeric antibody that specifically binds anepitope comprised by an amino acid sequence selected from the groupconsisting of an amino acid sequence substantially identical with anamino acid sequence as set forth in SEQ ID NO.:2, SEQ ID NO.:4, SEQ IDNO.:6 and SEQ ID NO.:8. The isolated polynucleotides optionally comprisea region that encodes a V_(H) region selected from the group consistingof an amino acid sequence substantially identical to the sequence as setforth in SEQ ID NO.:10, SEQ ID NO.:14, SEQ ID NO.:18 and SEQ ID NO.:28.The isolated polynucleotides comprise a region that encodes a V_(L)region selected from the group consisting of an amino acid sequencesubstantially identical to the sequence as set forth in SEQ ID NO.:12,SEQ ID NO.:16, SEQ ID NO.:20 and SEQ ID NO.:26. Further, the isolatedpolynucleotides comprise a region that encodes both a V_(H) and V_(L)region selected from the group consisting of a V_(H) region of SEQ IDNO.:10 and a V_(L) region of SEQ ID NO.:12; V_(H) region of SEQ IDNO.:14 and a V_(L) region of SEQ ID NO.:16; V_(H) region of SEQ IDNO.:18 and a V_(L) region of SEQ ID NO.:20; and V_(H) region of SEQ IDNO.:28 and a V_(L) region of SEQ ID NO.:26. In other aspects, thepolynucleotide is one selected from the group consisting of SEQ IDNO.:9, SEQ ID NO.:11, SEQ ID NO.:13, SEQ ID NO.:15, SEQ ID NO.:17, SEQID NO.:19, SEQ ID NO.:25 and SEQ ID NO.:27.

In accordance with yet another aspect of the disclosure there isprovided methods of purifying an antigen comprising a T. cruzi aminoacid sequence comprised by the amino acid sequences as set forth in SEQID NOs.:1, 3, 5 or 7, comprising contacting a sample suspected ofcontaining a T. cruzi polypeptide with an immunodiagnostic reagent, theimmunodiagnostic reagent comprising one or more antibodies, includingchimeric or recombinant antibodies, that are capable of specificallybinding to a T. cruzi protein, under conditions that allow formation ofantibody:antigen complexes, isolating the formed antibody:antigencomplexes and separating the antigen from the antibody. Optionally, theantibody, including chimeric and recombinant antibodies, binds to a T.cruzi polypeptide selected form the group consisting of FP3, Pep2, FP10,and FRA.

These and other features, aspects, objects, and embodiments of thedisclosure will become more apparent in the following detaileddescription in which reference is made to the appended drawings that areexemplary of such features, aspects, objects and embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 presents a diagrammatic structure of the chimeric (mouse-human)anti-T. cruzi epitope antibodies of the disclosure.

FIG. 2 depicts schematically the plasmid Chagas 12-392-150 Mu-Hu_pBJ,plasmid size: 9520 nucleotides. An ampicillin resistance gene ORF islocated at bases 60-917; an enhancer is located at bases 1551-2021; apromoter is located at bases 2023-2744; a heavy chain signal peptide islocated at bases 2772-2828; a V_(H) gene is located at bases 2829-3194;a human constant hgG1, z, non-a is located at bases 3195-4187; a SV40Poly A is located at bases 4219-4413; a SV40 promoter is located atbases 4684-5229; a murine DHFR is located at bases 5257-5820; a TK polyA is located at bases 5847-6213; an enhancer is located at bases6241-6711; a promoter is located at bases 6712-7433; a kappa signalpeptide is located at bases 7460-7525; a V_(L) gene is located at bases7526-7861; a human constant kappa is located at bases 7862-8185; a SV40Poly A is located at bases 8198-8392; and a pUC origin is located atbases 8759-9432 (complementary).

FIGS. 3A-C depicts the annotated, double-stranded polynucleotidesequence for VH and VL sequences (and flanking regions) cloned intoChagas 12-392-150 Mu-Hu_pBJ. FIG. 3A-B depicts the polynucleotidesequence (SEQ ID NOs.:21-22) for the Heavy chain signal peptide locatedat bases 2772-2828, VH gene sequences located at bases 2829-3194, andHuman Constant IgG1, z, non-a sequences located at bases 3195-4187. FIG.3C depicts the polynucleotide sequence (SEQ ID NOs.:23-24) for the Kappasignal peptide located at bases 7460-7525, the VL gene sequences locatedat bases 7526-7861, and the Human Constant kappa sequences located atbases 7862-8185.

FIG. 4 depicts schematically the plasmid Chagas 9-638 Mu-Hu_pBJ, plasmidsize: 9514 nucleotides. An ampicillin resistance gene ORF is located atbases 60-917; an enhancer is located at bases 1551-2021; a promoter islocated at bases 2023-2744; a heavy chain signal peptide is located atbases 2772-2828; a V_(H) gene is located at bases 2829-3188; a humanconstant hgG1, z, non-a is located at bases 3189-4181; a SV40 poly A islocated at bases 4213-4407; a SV40 promoter is located at bases4678-5223; a murine DHFR is located at bases 5251-5814; a TK poly A islocated at bases 5841-6207; an enhancer is located at bases 6235-6705; apromoter is located at bases 6706-7427; a kappa signal peptide islocated at bases 7454-7519; a V_(L) gene is located at bases 7520-7858;a human constant kappa is located at bases 7859-8179; a SV40 Poly A islocated at bases 8192-8386; and a pUC origin is located at bases8753-9426 (complementary).

FIG. 5 depicts schematically the plasmid Chagas 10-745 Mu-Hu_pBJ,plasmid size: 9514 nucleotides. An ampicillin resistance gene ORF islocated at bases 60-917; an enhancer is located at bases 1551-2021; apromoter is located at bases 2023-2744; a heavy chain signal peptide islocated at bases 2772-2828; a V_(H) gene is located at bases 2829-3188;a human constant IgG1, z, non-a is located at bases 3189-4181; a SV40Poly A is located at bases 4213-4407; a SV40 promoter is located atbases 4678-5223; a Murine DHFR is located at bases 5251-5814; a TK polyA is located at bases 5841-6207; an enhancer is located at bases6235-6705; a promoter is located at bases 6706-7427; a kappa signalpeptide is located at bases 7454-7519; a V_(L) gene is located at bases7520-7855; a human constant kappa is located at bases 7856-8179; a SV40poly A is located at bases 8192-8386; and a pUC origin bases 8753-9426(complementary).

DETAILED DESCRIPTION

The present disclosure provides, among other things, methods, assays andkits for detecting or quantifying Trypanosoma (Schizotrypanum) cruziantigens. In accordance with one embodiment of the present disclosure,recombinant antibodies of the disclosure, including chimeric antibodies,specifically bind to diagnostically relevant regions of T. cruziproteins and are thus suitable for use, for example, as diagnosticreagents for the detection of T. cruzi, and/or as standardizationreagents or positive control reagents in assays for the detection of T.cruzi.

The present disclosure also thus provides for an immunodiagnosticreagent comprising one or more recombinant antibodies, includingchimeric antibodies, wherein each antibody is capable of specificallybinding a diagnostically relevant region of a T. cruzi protein. Therecombinant antibodies can be, for example, chimeric antibodies,humanized antibodies, antibody fragments, and the like. In anotherembodiment, the immunodiagnostic reagent comprises two or morerecombinant antibodies, including chimeric antibodies. Optionally theantibodies used in the immunodiagnostic reagent are each specific for adifferent T. cruzi antigenic protein, such that the immunodiagnosticreagent is capable of detecting a plurality of T. cruzi antigens.Optionally, the immunodiagnostic reagent comprises at least one or more,or at least two or more, recombinant antibodies specific for T. cruziantigens selected from the group consisting of a recombinant antibodyspecific for Chagas FP3 antigen, a recombinant antibody specific forChagas FP6 antigen, a recombinant antibody specific for Chagas FP10antigen, and a recombinant antibody specific for Chagas FRA antigen. Inyet another embodiment, the antibody or antibodies of theimmunodiagnostic reagent are novel monoclonal antibodies produced byhybridoma cell lines and are specific for T. cruzi antigens selectedfrom the group consisting of a monoclonal antibody specific for ChagasFP3 antigen, a monoclonal antibody specific for Chagas FP6 antigen, amonoclonal antibody specific for Chagas FP10 antigen, and a monoclonalantibody specific for Chagas FRA antigen.

In one embodiment, the present disclosure provides for the use of theimmunodiagnostic reagent as a standardization reagent in a T. cruzidetection assay, for instance, in place of human sera. In this context,the immunodiagnostic reagent optionally can be used, for example, toevaluate and standardize the performance of current and future T. cruzidetection assays.

These and additional embodiments, features, aspects, illustrations, andexamples of the disclosure are further described in the sections whichfollow. Unless defined otherwise herein, all technical and scientificterms used herein have the same meaning as commonly understood by one ofordinary skill in the art to which this disclosure belongs.

A. Definitions

As used herein, the singular forms “a,” “an” and “the” include pluralreferents unless the context clearly dictates otherwise. For therecitation of numeric ranges herein, each intervening number therebetween with the same degree of precision is explicitly contemplated.For example, for the range 6-9, the numbers 7 and 8 are contemplated inaddition to 6 and 9, and for the range 6.0-7.0, the numbers 6.0, 6.1,6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9 and 7.0 are explicitlycontemplated.

a) About

As used herein, the term “about” refers to approximately a +/−10%variation from the stated value. It is to be understood that such avariation is always included in any given value provided herein, whetheror not it is specifically referred to.

b) Antibody

The term “antibody” (Ab) as used herein comprises single Abs directedagainst a TCA (an anti-TCA Ab), anti-TCA Ab compositions withpoly-epitope specificity, single chain anti-TCA Abs, and fragments ofanti-TCA Abs. A “monoclonal antibody” (mAb) is obtained from apopulation of substantially homogeneous Abs, i.e., the individual Abscomprising the population are identical except for possiblenaturally-occurring mutations that can be present in minor amounts.Exemplary Abs include polyclonal (pAb), monoclonal (mAb), humanized,bi-specific (bsAb), heteroconjugate Abs and dual-variable domainimmunoglobulins (DVD-Ig®) and derivatives of dual-variable domainimmunoglobulins (such as triple variable domains) (Dual-variable domainimmunoglobulins and methods for making them are described in Wu, C., etal., Nature Biotechnology, 25(11):1290-1297 (2007) and WO2001/058956;the contents of each of which are herein incorporated by reference).

c) Antibody Fragment

The term “antibody fragment” or “antibody fragments,” as used herein,refers to a portion of an intact antibody comprising the antigen bindingsite or variable region of the intact antibody, wherein the portion isfree of the constant heavy chain domains (i.e., C_(H)2, C_(H)3, andC_(H)4, depending on antibody isotype) of the Fc region of the intactantibody. Examples of antibody fragments include, but are not limitedto, Fab fragments, Fab′ fragments, Fab′-SH fragments, F(ab′)₂ fragments,Fv fragments, diabodies, single-chain Fv (scFv) molecules, single chainpolypeptides containing only one light chain variable domain, singlechain polypeptides containing the three CDRs of the light chain variabledomain, single chain polypeptides containing only one heavy chainvariable region, and single chain polypeptides containing the three CDRsof the heavy chain variable region.

d) Bifunctional Antibody

The term “bifunctional antibody,” as used herein, refers to an antibodythat comprises a first arm having a specificity for one antigenic siteand a second arm having a specificity for a different antigenic site,i.e., the bifunctional antibodies have a dual specificity.

e) Biological Sample

The term “biological sample” includes tissues, cells and biologicalfluids isolated from a subject, as well as tissues, cells and fluidspresent within a subject. Biological samples from a subject containpolypeptide molecules. Examples of biological samples include wholeblood, serum, plasma, interstitial fluid, saliva, ocular lens fluid,cerebral spinal fluid, sweat, urine, milk, ascites fluid, mucous, nasalfluid, sputum, synovial fluid, peritoneal fluid, vaginal fluid, menses,amniotic fluid and semen. Detection methods can be used to detect a TCAin a biological sample in vitro as well as in vivo. In vitro techniquesfor detection of a TCA include enzyme-linked immunosorbent assays(ELISAs), Western blots, immunoprecipitations and immunofluorescence.Furthermore, in vivo techniques for detecting a TCA include introducinginto a subject a labeled anti-TCA antibody. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques.

f) Binding Constants

The term “association rate constant”, “k_(on)” or “k_(a)” as usedinterchangeably herein, refers to the value indicating the binding rateof an antibody to its target antigen or the rate of complex formationbetween an antibody and antigen as shown by the equation below:Antibody(“Ab”)+Antigen(“Ag”)→Ab−Ag.

The term “dissociation rate constant”, “k_(off)” or “k_(a)” as usedinterchangeably herein, refers to the value indicating the dissociationrate of an antibody from its target antigen or separation of Ab−Agcomplex over time into free antibody and antigen as shown by theequation below:Ab+Ag←Ab−Ag.

Methods for determining association and dissociation rate constants arewell known in the art. Using fluorescence-based techniques offers highsensitivity and the ability to examine samples in physiological buffersat equilibrium. Other experimental approaches and instruments such as aBIAcore® (biomolecular interaction analysis) assay can be used (e.g.,instrument available from BIAcore International AB, a GE Healthcarecompany, Uppsala, Sweden). Additionally, a KinExA® (Kinetic ExclusionAssay) assay, available from Sapidyne Instruments (Boise, Id.) can alsobe used.

The term “equilibrium dissociation constant” or “K_(D)” as usedinterchangeably, herein, refers to the value obtained by dividing thedissociation rate (k_(off)) by the association rate (k_(on)). Theassociation rate, the dissociation rate and the equilibrium dissociationconstant are used to represent the binding affinity of an antibody to anantigen.

g) Chimeric Antibody

The term “chimeric antibody” (or “cAb”) as used herein, refers to apolypeptide comprising all or a part of the heavy and light chainvariable regions of an antibody from one host species linked to at leastpart of the antibody constant regions from another host species.

h) Corresponding to or Corresponds to

The terms “corresponding to” or “corresponds to” indicate that a nucleicacid sequence is identical to all or a portion of a reference nucleicacid sequence. The term “complementary to” is used herein to indicatethat the nucleic acid sequence is identical to all or a portion of thecomplementary strand of a reference nucleic acid sequence. Forillustration, the nucleic acid sequence “TATAC” corresponds to areference sequence “TATAC” and is complementary to a reference sequence“GTATA.”

Unless otherwise specified herein, all nucleic acid sequences arewritten in a 5′ to 3′ direction, and all amino acid sequences arewritten in an amino- to carboxy-terminus direction.

i) Derivatized Antibody

The term “derivatized antibody” as used herein refers to an antibody orantibody portion that is derivatized or linked to another functionalmolecule. For example, an antibody or antibody fragment can befunctionally linked, by chemical coupling, genetic fusion, ornon-covalent association, etc., to one or more molecules, such asanother antibody, a detectable agent, a cytotoxic agent, apharmaceutical agent, and a polypeptide that can mediate association ofthe antibody or antibody portion with another molecule, such as astreptavidin core region or a polyhistidine tag. One type of derivatizedantibody is produced by cross-linking two or more antibodies. Suitablecross-linkers include those that are hetero-bifunctional (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homo-bifunctional(e.g., disuccinimidyl suberate). Such linkers are available from PierceChemical Company (Rockford, Ill.).

j) Detectable Label

The term, “detectable labels”, as used herein, include molecules ormoieties that can be detected directly or indirectly. Furthermore, theseagents can be derivatized with antibodies and include fluorescentcompounds. Classes of labels include fluorescent, luminescent,bioluminescent, and radioactive materials, enzymes and prostheticgroups. Useful labels include horseradish peroxidase, alkalinephosphatase, β-galactosidase, acetylcholinesterase, streptavidin/biotin,avidin/biotin, umbelliferone, fluorescein, fluorescein isothiocyanate,rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride,phycoerythrin, luminol, luciferase, luciferin, aequorin, and ¹²⁵I, ¹³¹I,³⁵S or ³H.

k) Diagnostically Relevant

The term “diagnostically relevant” as used herein with reference to aregion of a T. cruzi protein refers to a region of the protein thedetection of which, either alone or in combination with otherdiagnostically relevant regions of Chagas, allows detection of T. cruzi.Examples of diagnostically relevant regions include immunodominantregions known in the art and regions such as those described herein.

l) Epitope, Epitopes or Epitopes of Interest

As used herein, the term “epitope”, “epitopes” or “epitopes of interest”refer to a site(s) on any molecule that is recognized and is capable ofbinding to a complementary site(s) on its specific binding partner. Themolecule and specific binding partner are part of a specific bindingpair. For example, an epitope can be a polypeptide, protein, hapten,carbohydrate antigen (such as, but not limited to, glycolipids,glycoproteins or lipopolysaccharides) or polysaccharide and its specificbinding partner, can be, but is not limited to, an antibody. Typicallyan epitope is contained within a larger antigenic fragment (i.e., regionor fragment capable of binding an antibody) and refers to the preciseresidues known to contact the specific binding partner. It is possiblefor an antigenic fragment to contain more than one epitope.

m) Humanized Antibody

The term “humanized antibody,” as used herein, refers to a polypeptidecomprising a modified variable region of a human antibody wherein aportion of the variable region has been substituted by the correspondingsequence from a non-human species and wherein the modified variableregion is linked to at least part of the constant region of a humanantibody. In one embodiment, the portion of the variable region is allor a part of the complementarity determining regions (CDRs). The termalso includes hybrid antibodies produced by splicing a variable regionor one or more CDRs of a non-human antibody with a heterologousprotein(s), regardless of species of origin, type of protein,immunoglobulin class or subclass designation, so long as the hybridantibodies exhibit the desired biological activity (i.e., the ability tospecifically bind a T. cruzi antigenic protein).

n) Isolated or Purified

The term “isolated” or “purified”, when referring to a molecule, refersto a molecule that has been identified and separated and/or recoveredfrom a component of its natural environment. Contaminant components ofits natural environment are materials that interfere with diagnostic ortherapeutic use. The term “isolated” or “purified” polypeptide orbiologically active fragment (such as an Fab fragment) as used hereinrefers to a polypeptide or biologically active fragment that isseparated and/or recovered from a component of its environment.Contaminant components include materials that would typically interferewith diagnostic uses for the polypeptide, and can include enzymes,hormones, and other polypeptideaceous or non-polypeptideaceousmaterials. To be substantially isolated, preparations having less thanabout 30% by dry weight of contaminants (i.e., from about 0.01% to about30%), usually less than about 20% (i.e., from about 0.01% to about 20%),less than about 10% (i.e., from about 0.01% to about 10%), and moreoften, less than about 5% (i.e., from about 0.01% to about 5%)contaminants. An isolated, recombinantly-produced TCA, V_(L) or V_(H) orbiologically active portion is desirably substantially free of culturemedium, i.e., culture medium represents less than about 20%, about 10%,or about 5% of the volume of the TCA, V_(L) or V_(H) preparation.Therefore, an “isolated antibody” as used herein refers to an antibodythat is substantially free of other antibodies having differentantigenic specificities. An isolated antibody that specifically binds aT. cruzi epitope can, however, have cross-reactivity to other T. cruziantigens, such as, for example, an antibody that bind the Pep2 epitope,found on the Chagas polypeptides Tcf and FP6.

o) Quality Control Reagents

As described herein, immunoassays incorporate “quality control reagents”that include but are not limited to, e.g., calibrators, controls, andsensitivity panels. A “calibrator” or “standard” typically is used(e.g., one or more, or a plurality) in order to establish calibration(standard) curves for interpolation of antibody concentration.Optionally, a single calibrator can be used near the positive/negativecutoff. Multiple calibrators (i.e., more than one calibrator or avarying amount of calibrator(s)) can be used in conjunction so as tocomprise a “sensitivity panel. A “positive control” is used to establishassay performance characteristics and is a useful indicator of theintegrity of the reagents (e.g., antigens).

p) Recombinant Antibody or Recombinant Antibodies

The term “recombinant antibody” or “recombinant antibodies,” as usedherein, refers to an antibody prepared by one or more steps includingcloning nucleic acid sequences encoding all or a part of one or moremonoclonal antibodies into an appropriate expression vector byrecombinant techniques and subsequently expressing the antibody in anappropriate host cell. The term thus includes, but is not limited to,recombinantly-produced antibodies that are monoclonal antibodies,antibody fragments including fragments of monoclonal antibodies,chimeric antibodies, humanized antibodies (fully or partiallyhumanized), multispecific or multivalent structures formed from antibodyfragments (including tetravalent IgG-like molecules termeddual-variable-domain immunoglobulin, DVD-Ig®), and bifunctionalantibodies.

q) Specific or Specificity

As used herein, “specific” or “specificity” in the context of aninteraction between members of a specific binding pair (e.g., an antigenand antibody) refers to the selective reactivity of the interaction. Thephrase “specifically binds to” and analogous terms thereof refer to theability of antibodies to specifically bind to a T. cruzi protein and notspecifically bind to other entities. Antibodies or antibody fragmentsthat specifically bind to a T. cruzi protein can be identified, forexample, by diagnostic immunoassays (e.g., radioimmunoassays (“RIA”) andenzyme-linked immunosorbent assays (“ELISAs”) (See, for example, Paul,ed., Fundamental Immunology, 2nd ed., Raven Press, New York, pages332-336 (1989)), BIAcore® (biomolecular interaction analysis, instrumentavailable from BIAcore International AB, Uppsala, Sweden), KinExA®(Kinetic Exclusion Assay, available from Sapidyne Instruments (Boise,Id.)) or other techniques known to those of skill in the art.

r) Substantially Identical

The term “substantially identical,” as used herein in relation to anucleic acid or amino acid sequence indicates that, when optimallyaligned, for example using the methods described below, the nucleic acidor amino acid sequence shares at least about 70% (e.g., from about 70%to about 100%), at least about 75% (e.g., from about 75% to about 100%),at least about 80% (e.g., from about 80% to about 100%), at least about85% (e.g., from about 85% to about 100%), at least about 90% (e.g., fromabout 90% to about 100%), at least about 95% (e.g., from about 95% toabout 100%), at least about 96% (e.g., from about 96% to about 100%), atleast about 97% (e.g., from about 97% to about 100%), at least about 98%(e.g., from about 98% to about 100%), or at least about 99% (e.g., fromabout 99% to about 100%) sequence identity with a defined second nucleicacid or amino acid sequence (or “reference sequence”). “Substantialidentity” can be used to refer to various types and lengths of sequence,such as full-length sequence, epitopes or immunogenic peptides,functional domains, coding and/or regulatory sequences, exons, introns,promoters, and genomic sequences. Percent identity between two aminoacid or nucleic acid sequences can be determined in various ways thatare within the skill of a worker in the art, for example, using publiclyavailable computer software such as Smith Waterman Alignment (Smith, T.F. and M. S. Waterman (1981) J Mol Biol 147:195-7); “BestFit” (Smith andWaterman, Advances in Applied Mathematics, 482-489 (1981)) asincorporated into GeneMatcher Plus™, Schwarz and Dayhof (1979) Atlas ofProtein Sequence and Structure, Dayhof, M. O., Ed pp 353-358; BLASTprogram (Basic Local Alignment Search Tool (Altschul, S. F., W. Gish, etal. (1990) J Mol Biol 215: 403-10), and variations thereof includingBLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL,and Megalign (DNASTAR) software. In addition, those skilled in the artcan determine appropriate parameters for measuring alignment, includingalgorithms needed to achieve maximal alignment over the length of thesequences being compared. In general, for amino acid sequences, thelength of comparison sequences is at least about 10 amino acids. Oneskilled in the art understands that the actual length depends on theoverall length of the sequences being compared and can be at least about20, at least about 30, at least about 40, at least about 50, at leastabout 60, at least about 70, at least about 80, at least about 90, atleast about 100, at least about 110, at least about 120, at least about130, at least about 140, at least about 150, at least about 200, atleast about 250, at least about 300, or at least about 350 amino acids,or it can be the full-length of the amino acid sequence. For nucleicacids, the length of comparison sequences is generally at least about 25nucleotides, but can be at least about 50, at least about 100, at leastabout 125, at least about 150, at least about 200, at least about 250,at least about 300, at least about 350, at least about 400, at leastabout 450, at least about 500, at least about 550, at least about 600,at least about 650, at least about 700, at least about 800, at leastabout 900, or at least about 1000 nucleotides, or it can be thefull-length of the nucleic acid sequence.

s) Surface Plasmon Resonance

The term “surface plasmon resonance” as used herein refers to an opticalphenomenon that allows for the analysis of real-time biospecificinteractions by detecting alterations in protein concentrations within abiosensor matrix, for example using the BIACORE® system (Biacore (GEHealthcare)) (Johnsson, B., et al. 1991. Immobilization of proteins to acarboxymethyldextran-modified gold surface for biospecific interactionanalysis in surface plasmon resonance sensors. Anal Biochem. 198:268-77;Johnsson, B., et al. 1995. Comparison of methods for immobilization tocarboxymethyl dextran sensor surfaces by analysis of the specificactivity of monoclonal antibodies. J Mol Recognit. 8:125-31; Jonsson,U., et al. 1993. Introducing a biosensor based technology for real-timebiospecific interaction analysis. Ann Biol Clin (Paris). 51:19-26).

t) TCA

The abbreviation “TCA,” as used herein, means “T. cruzi antigen.” FP3,Pep2, TcF, FP6, and FP10 refer to TCAs and are further defined below.Other abbreviations are defined as they are introduced.

The terminology used herein is for the purpose of describing particularembodiments only and is not otherwise intended to be limiting.

B. Anti-T. Cruzi Antibodies and Cell Lines Producing Same

The present disclosure provides, among other things, novel antibodies,cell lines producing these antibodies, and methods of making theseantibodies. These antibodies bind various T. cruzi antigens (TCAs) andinclude those contained in the FP3, Pep2 (TcF, FP6) and FP10polypeptides, and can be used as mAbs, such as mouse mAbs, dual-variabledomain immunoglobulins (DVD-Ig®) or as chimeric antibodies, such asmouse-human (Mu-Hu) chimeras. These antibodies are useful as positivecontrols in immunoassays. Furthermore, the antibodies can be used topurify T. cruzi polypeptides that harbor the TCAs. Examples ofantibodies and cell lines of the present disclosure are presented belowin Table 1.

TABLE 1 T. cruzi Antigens and antibody-producing cell lines summary¹Hybridoma cell line CHO cell line ATCC ATCC Antigen Deposit* Deposit*Antigen Cell Line [Deposit Cell Line [Deposit Name Name Laboratory NameDate] Name Laboratory Name Date] FP3 HBFP3 Chagas FP3 12-392- PTA-8139CHOFP3 Chagas FP3 12-392- PTA-8136 150-110 [Jan. 24, 2007]150CHO2580-104 [Jan. 24, 2007] Pep2 (TcF, HBPep2 Chagas 9-638-132-PTA-8137 CHOPep2 Chagas Pep2 9-638-1928 PTA-8138 FP6) 115 [Jan. 24,2007] [Jan. 24, 2007] FP10 HBF10 Chagas 10-745-140 PTA-8141 CHOFP10Chagas FP10 10-745-3796 PTA-8140 [Jan. 24, 2007] [Jan. 24, 2007]¹Another hybridoma cell line, laboratory name Chagas 8-367-171 andproducing a mAb that binds recombinant FRA antigen, is deposited asPTA-8142 (also deposited on Jan. 24, 2007). *All cell line deposits weremade under the Budapest Treaty on the International Recognition of theDeposit of Microorganisms for the Purposes of Patent Procedure (BudapestTreaty) of Apr. 28, 1977 and amended on Sep. 26, 1980. American TypeCulture Collection (ATCC); P.O. Box 1549; Manassas, VA 20108; USA.

Further examples of antibodies of the present disclosure are antibodiesthat:

(a) that specifically binds to a diagnostically relevant region of a T.cruzi polypeptide, wherein the T. cruzi polypeptide is FRA and furtherwherein said antibody has at last one binding constant selected from thegroup consisting of: an association rate constant (k_(a)) between about7.0×10⁵ Ms to about 7.0×10⁶M⁻¹ s⁻¹, an dissociation rate constant(k_(d)) between about 4.0×10⁻³ s⁻¹ to about 3.0×10⁻¹ s⁻¹ and anequilibrium dissociation constant (K_(D)) between about 5.7×10⁻¹⁰ M toabout 4.3×10⁻⁷ M;

(b) that specifically binds to a diagnostically relevant region of a T.cruzi polypeptide, wherein the T. cruzi polypeptide is Pep2 and furtherwherein said antibody has at least one binding constant selected fromthe group consisting of: an association rate constant (k_(a)) betweenabout 1.0×10⁶M⁻¹ s⁻¹ to about 8.0×10⁶M⁻¹ s⁻¹; an dissociation rateconstant (k_(d)) between about 6.0×10⁻³ s⁻¹ to about 4.0×10⁻² s¹ and anequilibrium dissociation constant (K_(D)) between about 7.5×10⁻¹⁰ M toabout 4.0×10⁻⁸ M;

(c) that specifically binds to a diagnostically relevant region of a T.cruzi polypeptide, wherein the T. cruzi polypeptide is FP10 and furtherwherein said antibody has at least one binding constant selected fromthe group consisting of: (a) an association rate constant (k_(a))between about 5.0×10⁴M⁻¹ s⁻¹ to about 3.0×10⁵M⁻¹ s⁻¹: (b) andissociation rate constant (k_(d)) between about 1.0×10⁻⁴ s⁻¹ to about8.0×10⁻⁴ s⁻¹; and (c) an equilibrium dissociation constant (K_(D))between about 3.3×10⁻¹⁰ M to about 1.6×10⁻⁸M;

(d) that specifically binds to a diagnostically relevant region of a T.cruzi polypeptide, wherein the T. cruzi polypeptide is FP3 and furtherwherein said antibody has at least one binding constant selected fromthe group consisting of: an association rate constant (k_(a)) betweenabout 2.0×10⁵M⁻¹ s⁻¹ to about 6.0×10⁶M⁻¹ s⁻¹; an dissociation rateconstant (k_(d)) between about 2.0×10⁻⁵ s⁻¹ to about 8.0×10⁻⁴ s⁻¹; andan equilibrium dissociation constant (K_(D)) between about 3.3×10⁻¹²M toabout 4.0×10⁻⁹ M; and

(e) any combinations of (a)-(d).

To make the anti-T. cruzi antibodies and cell lines producing theseantibodies as further described herein, generally a two-step process wasfollowed: (1) hybridoma cell lines were developed that producedmonoclonal antibodies that specifically bound to the antigens ofinterest—the T. cruzi epitopes (TCAs); and (2) chimeric antibodies wereengineered using recombinant technologies, and then mammalian expressioncell lines were used to produce the engineered antibodies. In thissecond part, after identifying hybridoma cell lines that secreted thedesired mAbs, mRNA was isolated from these cells and the antibody genesequences were identified. The variable light (V_(L)) and variable heavy(V_(H)) polynucleotide sequences were then cloned into pBOS vectors(supplying the human antibody sequences) that were then co-transfectedin a transient expression system to confirm that the resulting chimericantibodies were functional. Upon confirmation, the V_(L) sequences weresub-cloned into the pJV plasmid, and the V_(H) sequences into the pBVplasmid; these vectors where then used to construct a stable pBJexpression vector. CHO cells were then transfected with pBJ,transfectants selected, and the secreted antibodies tested again,allowing for industrial scale production. Thus, the mouse V_(H) andV_(L) regions were combined with human constant chain (CH) and constantlight chain (CL) regions to create exemplars of the chimeric antibodiesof the disclosure. Therefore, the chimeric antibodies retain the mousemAb functional specificity and affinity for the TCAs, but react inantibody assays that are designed to detect human immunoglobulin (Ig).In one embodiment, the disclosure is directed to monoclonal antibodies(mAbs) that specifically bind the TCAs FP3, Pep2 (FP6/Tcf), FP10 andFRA. Mice are individually immunized with the FP3, Pep2, FP10 or FRArecombinant antigens, antibody-producing mice are identified andeuthanized, spleen cells are harvested and fused with myeloma cells, andmAb producing hybridoma cell lines are isolated.

C. Immunodiagnostic Reagent

The immunodiagnostic reagent of the present disclosure comprises one ormore antibodies described herein (See, for example, Sections B and Eherein). For example the antibodies comprising the immunodiagnosticreagent can include recombinant antibodies, which also herein includerecombinant chimeric antibodies, that specifically bind to adiagnostically relevant region of a T. cruzi protein. Therefore, in oneembodiment, the immunodiagnostic reagent provided by the presentdisclosure comprises a single antibody capable of specifically binding adiagnostically relevant region of a T. cruzi protein. In otherembodiments, the immunodiagnostic reagent provided by the presentdisclosure comprises a single chimeric antibody capable of specificallybinding a diagnostically relevant region of a T. cruzi protein. In otherembodiments, the immunodiagnostic reagent comprises a plurality ofantibodies, which can include one or more recombinant antibodies, suchas a recombinant chimeric antibody, each capable of specifically bindinga diagnostically relevant region of a T. cruzi protein (e.g., either thesame region, or a different region). One or more of the plurality ofchimeric antibodies can be capable of specifically binding adiagnostically relevant region of the same T. cruzi protein.Alternatively, each of the plurality of chimeric antibodies canspecifically bind a diagnostically relevant region of a different T.cruzi protein.

In one embodiment, of the present disclosure, the immunodiagnosticreagent is capable of detecting a plurality of T. cruzi antigens andoptionally comprises two or more recombinant antibodies, each capable ofspecifically binding a different T. cruzi antigenic protein. In afurther embodiment, the immunodiagnostic reagent optionally comprisesthree or more recombinant antibodies, each capable of specificallybinding a different T. cruzi antigenic protein. In another embodiment,the immunodiagnostic reagent optionally comprises four or morerecombinant antibodies, each capable of specifically binding a differentT. cruzi antigenic protein.

The recombinant antibodies comprised by the immunodiagnostic reagent canoptionally be modified, for example, for detection purposes, forimmobilization onto a solid support, to improve stability and/or toimprove pharmacokinetic properties, or by other means such as is knownin the art.

D. T. Cruzi Antigens

T. cruzi is a complex organism, with a complex life cycle. However,important antigens have been identified that are useful for thediagnostic detection of the parasite.

The FP3 antigen (Kirchhoff, L. V., and K. Otsu. U.S. Patent ApplicationPublication No. 2004/0132077. 2004) is a recombinant protein thecorresponds essentially to the combination of T. cruzi Ag15 (Otsu, K.,et al. 1993. Interruption of a Trypanosoma cruzi gene encoding a proteincontaining 14-amino acid repeats by targeted insertion of the neomycinphosphotransferase gene. Mol Biochem Parasitol. 57:317-30) and T. cruziProtein C, the latter being a flagellar calcium binding protein(Gonzalez, A., et al. 1985. Apparent generation of a segmented mRNA fromtwo separate tandem gene families in Trypanosoma cruzi. Nucleic AcidsRes. 13:5789-804). The polynucleotide sequence (SEQ ID NO.:1) and thepolypeptide sequence (SEQ ID NO.:2) are shown below in Tables 2 and 3,respectively. The amino acid sequences specific to T. cruzi 14-aminoacid repeats are underlined in Table 3, those amino acids correspondingto T. cruzi Protein A are in bold in Table 3, those amino acidscorresponding to Protein B are in italics in Table 3 and those aminoacids corresponding to Protein C are twice underscored in Table 3.

TABLE 2 FP3 polynucleotide sequence (SEQ ID NO.: 1)ATGGCCCAGC TCCAACAGGC AGAAAATAAT ATCACTAATT  CCAAAAAAGA AATGACAAAG CTACGAGAAA AAGTGAAAAAGGCCGAGAAA GAAAAATTGG ACGCCATTAA CCGGGCAACCAAGCTGGAAG AGGAACGAAA CCAAGCGTAC AAAGCAGCAC ACAAGGCAGA GGAGGAAAAG GCTAAAACAT TTCAACGCCTTATAACATTT GAGTCGGAAA ATATTAACTT AAAGAAAAGGCCAAATGACG CAGTTTCAAA TCGGGATAAG AAAAAAAATT CTGAAACCGC AAAAACTGAC GAAGTAGAGA AACAGAGGGCGGCTGAGGCT GCCAAGGCCG TGGAGACGGA GAAGCAGAGGGCAGCTGAGG CCACGAAGGT TGCCGAAGCG GAGAAGCGGA AGGCAGCTGA GGCCGCCAAG GCCGTGGAGA CGGAGAAGCAGAGGGCAGCT GAAGCCACGA AGGTTGCCGA AGCGGAGAAGCAGAAGGCAG CTGAGGCCGC CAAGGCCGTG GAGACGGAGA AGCAGAGGGC AGCTGAAGCC ACGAAGGTTG CCGAAGCGGAGAAGCAGAGG GCAGCTGAAG CCATGAAGGT TGCCGAAGCGGAGAAGCAGA AGGCAGCTGA GGCCGCCAAG GCCGTGGAGA CGGAGAAGCA GAGGGCAGCT GAAGCCACGA AGGTTGCCGAAGCGGAGAAG CAGAAGGCAG CTGAGGCCGC CAAGGCCGTGGAGACGGAGA AGCAGAGGGC AGCTGAAGCC ACGAAGGTTG CCGAAGCGGA GAAGCAGAAG GCAGCTGAGG CCGCCAAGGCCGTGGAGACG GAGAAGCAGA GGGCAGCTGA AGCCACGAAGGTTGCCGAAG CGGAGAAGGA TATCGATCCC ATGGGTGCTT GTGGGTCGAA GGACTCGACG AGCGACAAGG GGTTGGCGAGCGATAAGGAC GGCAAGAACG CCAAGGACCG CAAGGAAGCGTGGGAGCGCA TTCGCCAGGC GATTCCTCGT GAGAAGACCG CCGAGGCAAA ACAGCGCCGC ATCGAGCTCT TCAAGAAGTTCGACAAGAAC GAGACCGGGA AGCTGTGCTA CGATGAGGTGCACAGCGGCT GCCTCGAGGT GCTGAAGTTG GACGAGTTCA CGCCGCGAGT GCGCGACATC ACGAAGCGTG CATTCGACAAGGCGAGGGCC CTGGGCAGCA AGCTGGAGAA CAAGGGCTCCGAGGACTTTG TTGAATTTCT GGAGTTCCGT CTGATGCTGT GCTACATCTA CGACTTCTTC GAGCTGACGG TGATGTTCGACGAGATTGAC GCCTCCGGCA ACATGCTGGT TGACGAGGAGGAGTTCAAGC GCGCCGTGCC CAGGCTTGAG GCGTGGGGCG CCAAGGTCGA GGATCCCGCG GCGCTGTTCA AGGAGCTCGATAAGAACGGC ACTGGGTCCG TGACGTTCGA CGAGTTTGCTGCGTGGGCTT CTGCAGTCAA ACTGGACGCC GACGGCGACC CGGACAACGT GCCGGAGAGC CCGAGACCGA TGGGAATC

TABLE 3 FP3 polypeptide sequence (SEQ ID NO.: 2)MAQLQQAENN ITNSKKEMTK LREKVKKAEK EKLDAINRATKLEEERNQAY KAAHKAEEEK AKTFQRLITF ESENINLKKR PNDAVSNRDK KKNSETAKTD EVEKQRAAEAAKAVET EKQRAAEATKVAEA EKRKAAEAAKAVET EKQRAAEATKVAEAEKQKAAEAAKAVET EKQRAAEATKVAEA EKQRAAEAMKVAEA EKQKAAEAAKAVETEKQRAAEATKVAEA EKQKAAEAAKAVET EKQRAAEATKVAEA EKQKAAEAAKAVETEKQRAAEATKVAEA EKDIDP MGACGSKDST SDKGLASDKD GKNAKDRKEA WERIRQAIPREKTAEAKQRR IELFKKFDKN ETGKLCYDEV HSGCLEVLKLDEFTPRVRDI TKRAFDKARA LGSKLENKGS EDFVEFLEFRLMLCYIYDFF ELTVMFDEID ASGNMLVDEE EFKRAVPRLEAWGAKVEDPA ALFKELDKNG TGSVTFDEFA AWASAVKLDA DGDPDNVPES PRPMGI

The Pep2 antigen (Kirchhoff and Otsu, 2004) is a recombinant protein ofrepeated sequences of T. cruzi. FP6 and Tcf, T. cruzi polypeptides, bothhave the Pep2 antigen. The polynucleotide sequence (SEQ ID NO.:3) andpolypeptide sequence (SEQ ID NO.:4) is shown in Tables 4 and 5,respectively.

TABLE 4 Pep2 polynucleotide sequence (SEQ ID NO.: 3)GGTGACAAAC CATCACCATT TGGACAGGCC GCAGCAGGTG ACAAACCATC ACCATTTGGA CAGGCC

TABLE 5 Pep2 polypeptide sequence (SEQ ID NO.: 4)GDKPSPFGQA AAGDKPSPFG QA

The FP10 antigen (Kirchhoff and Otsu, 2004) is another recombinantprotein of repeated sequences of T. cruzi. Its polynucleotide (SEQ IDNO.: 5) and polypeptide (SEQ ID NO.:6) sequences are shown below inTables 6 and 7, respectively. The amino acid sequence of the I-domain isunderlined in Table 7, the amino acid sequence of the J-domain is initalics in Table 7, the amino acid sequence of the K-domain is in boldin Table 7 and the amino acid sequence of the L-domain are twiceunderscored in Table 7.

TABLE 6 FP10 polynucleotide sequence (SEQ ID NO.: 5)GATCCAACGT ATCGTTTTGC AAACCACGCG TTCACGCTGGTGGCGTCGGT GACGATTCAC GAGGTTCCGA GCGTCGCGAGTCCTTTGCTG GGTGCGAGCC TGGACTCTTC TGGTGGCAAAAAACTCCTGG GGCTCTCGTA CGACGAGAAG CACCAGTGGCAGCCAATATA CGGATCAACG CCGGTGACGC CGACCGGATCGTGGGAGATG GGTAAGAGGT ACCACGTGGT TCTTACGATGGCGAATAAAA TTGGCTCCGT GTACATTGAT GGAGAACCTCTGGAGGGTTC AGGGCAGACC GTTGTGCCAG ACGAGAGGACGCCTGACATC TCCCACTTCT ACGTTGGCGG GTATGGAAGGAGTGATATGC CAACCATAAG CCACGTGACG GTGAATAATGTTCTTCTTTA CAACCGTCAG CTGAATGCCG AGGAGATCAGGACCTTGTTC TTGAGCCAGG ACCTGATTGG CACGGAAGCACACATGGGCA GCAGCAGCGG CAGCAGTGCC CACGGTACGCCCTCGATTCC CGTTGACAGC AGTGCCCACG GTACACCCTCGACTCCCGTT GACAGCAGTG CCCACGGTAC GCCCTCGACTCCCGTTGACA GCAGTGCCCA CGGTACACCC TCGACTCCCGTTGACAGCAG TGCCCACGGT ACACCCTCGA CTCCCGTTGACAGCAGTGCC CACGGTAAGC CCTCGACTCC CGCTGACAGCAGTGCCCACA GTACGCCCTC GACTCCCGCT GACAGCAGTGCCCACAGTAC GCCCTCAATT CCCGCTGACA GCAGTGCCCACAGTACGCCC TCAGCTCCCG CTGACAACGG CGCCAATGGTACGGTTTTGA TTTTGTCGAC TCATGACGCG TACAGGCCCGTTGATCCCTC GGCGTACAAG CGCGCCTTGC CGCAGGAAGAGCAAGAGGAT GTGGGGCCGC GCCACGTTGA TCCCGACCACTTCCGCTCGA CCTCGACGAC TCATGACGCG TACAGGCCCGTTGATCCCTC GGCGTACAAG CGCGCCTTGC CGCAGGAAGAGCAAGAGGAT GTGGGGCCGC GCCACGTTGA TCCCGACCACTTCCGCTCGA CGACTCATGA CGCGTACAGG CCCGTTGATCCCTCGGCGTA CAAGCGCGCC TTGCCGCAGG AAGAGCAAGAGGATGTGGGG CCGCGCCACG TTGATCCCGA CCACTTCCGCTCGACCTCGA CGACTCATGA CGCGTACAGG CCCGTTGATCCCTCGGCGTA CAAGCGCGCC TTGCCGCAGG AAGAGCAAGAGGATGTGGGG CCGCGCCACG TTGATCCCGA CCACTTCCGCTCGACCTCGA CGACTCATGA CGCGTACAGG CCCGTTGATCCCTCGGCGTA CAAGCGCGCC TTGCCGCAGG AAGAGCAAGAGGATGTGGGG CCGCGCCACG TTGATCCCGA CCACTTCCGCTCGACGACTC ATGACGCGTA CAGGCCCGTT GATCCCTCGGCGTACAAGCG CGCCTTGCCG CAGGAAGAGC AAGAGGATGTGGGGCCGCGC CACGTTGATC CCGACCACTT CCGCTCG

TABLE 7 FP10 polypeptide sequence (SEQ ID NO.: 6)DPTYRFANHA FTLVASVTIH EVPSVASPLL GASLDSSGGKKLLGLSYDEK HQWQPIYGST PVTPTGSWEM GKRYHVVLTMANKIGSVYID GEPLEGSGQT VVPDERTPDI SHFYVGGYGRSDMPTISHVT VNNVLLYNRQ LNAEEIRTLF LSQDLIGTEA HMGSSSG SSAHGTPSIPVDSSAHGTPSTPVD SSAHGTPSTPVD SSAHGTPSTPVD SSAHGTPSTPVD SSAHGKPSTPADSSAHSTPSTPAD SSAHSTPSIPAD SSAHSTPSAPAD NGANGTV LILSTHDAYR PVDPSAYKRA LPQEEQEDVGPRHVDPDHFR STSTTHDAYR PVDPSAYKRA LPQEEQEDVGPRHVDPDHFR STTHDAYRPV DPSAYKRALP QEEQEDVGPRHVDPDHFRST STTHDAYRPV DPSAYKRALP QEEQEDVGPRHVDPDHFRST STTHDAYRPV DPSAYKRALP QEEQEDVGPRHVDPDHFRST THDAYRPVDP SAYKRALPQE EQEDVGPRHV DPDHFRS

The FRA antigen is a flagellar repetitive protein sequence (Lafaille, J.J., etc. 1989. Structure and expression of two Trypanosoma cruzi genesencoding antigenic proteins bearing repetitive epitopes. Mol BiochemParasitol. 35:127-36), GenBank Accession J04015, is shown below in Table8 (polynucleotide sequence, SEQ ID NO.:7) and 9 (polypeptide sequence;SEQ ID NO.:8).

TABLE 8 FRA polynucleotide sequence (SEQ ID NO.: 7)ATGGAGTCAG GAGCGTCAGA TCAGCTGCTC GAGAAGGACCCGCGTCAGGA ACGCGAAGGA GATTGCTGCG CTTGAGGAGAGTCATGAATG CCCGCGTCAT CAGGAGCTGG CGCGCGAGAAGAAGCTTGCC GACCGCGCGT TCCTTGACTC AGAAGCCGGAGCGCGTGCCG CTGGCTGACG TGCCGCTCGA CGACGATCAG CGACTTTGTT GCG

TABLE 9 FRA polypeptide sequence (SEQ ID NO.: 8)MEQERRQLLE KDPRRNAKEI AALEESMNAR AQELAREKKLADRAFLDQKP ERVPLADVPL DDDSDFVA

The TCAs of SEQ ID NOs.:2, 4, 6 and 8 can be either synthesized in vitroor expressed recombinantly from the polynucleotide sequences, such asthose substantially similar to SEQ ID NOs.:1, 3, 5 and 7. Because ofredundancy in the genetic code and the ability for the polypeptides ofSEQ ID NOs.:2, 4, 6 and 8 to tolerate substitutions, the sequences neednot be identical to practice the disclosure. Polynucleotide andpolypeptide sequence identities can range from about 70% to about 100%(especially from about from about 90% to about 97%), such as about 70%,about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%,about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about98%, about 99% and of course, about 100%.

The TCAs can be readily synthesized in vitro using polypeptidechemistry. For example, polypeptide synthesis can be carried out in astepwise manner on a solid phase support using an automated polypeptidesynthesizer, such as a Rainin Symphony Peptide Synthesizer, AdvancedChemtech Peptide Synthesizer, Argonaut Parallel Synthesis System, or anApplied Biosystems Peptide Synthesizer. The peptide synthesizerinstrument combines the Fmoc chemistry with HOBt/HBTU/DIEA activation toperform solid-phase peptide synthesis.

Synthesis starts with the C-terminal amino acid, wherein the carboxylterminus is covalently linked to an insoluble polymer support resin.Useful resins can load 0.1 mmol to 0.7 mmol of C-terminal amino acid pergram of resin; display resistance to the various solvents and chemicalsused during a typical synthetic cycle, such as dichloromethane (DCM),N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP),N,N-dimethylamine (DMA), 1-Hydroxybenzotriazole (HOBt),2-(1-H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), N,N-di-isopropylethylamine (DIEA), methanol (MeOH), or water;and be amenable to continuous flow or batch synthesis applications.Examples of useful resins include p-Benzyloxybenzyl Alcohol resin (HMPresin), PEG co-Merrifield resin, NovaSyn TGA® resin (Novabiochem),4-sulfamylbutyryl AM resin, and CLEAR amide resin. Amino acid-coupledresins are commercially available from a number of different sources,although such coupled resins can also be prepared in the lab.

The N-terminus of the resin-coupled amino acid (or polypeptide) ischemically-protected by a 9-flourenylmethloxycarbonyl (Fmoc) group thatis removed prior to the addition of the next N-terminal amino acidreactant. The Fmoc group is a base labile protecting group that iseasily removed by concentrated solutions of amines, such as 20-55%piperidine, in a suitable solvent, such as NMP or DMF. Other usefulamines for Fmoc deprotection include tris (2-aminoethyl) amine,4-(aminomethyl)piperidine, tetrabutylammonium fluoride, and1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). Complete removal of the Fmocgroup from the N-terminus is important so that all resin-coupledpolypeptide chains effectively participate in each coupling cycle;otherwise, polypeptide chains of heterogeneous length and sequence willresult. Following base-catalyzed removal of the Fmoc group, the resin isextensively washed with a suitable buffer to remove the base catalyst.

The side chains of many amino acids contain chemically reactive groups,such as amines, alcohols, or thiols. These side chains must beadditionally protected to prevent undesired side-reactions during thecoupling step. Side chain protecting groups that are base-stable, morepreferably, both base-stabile and acid-labile are most useful. Table 10provides an exemplary set of side chain protection groups for thiscategory of amino acids.

TABLE 10 Side chain protection reagents Side chain protection Amino acidt-butyl ether Ser, Thr, Tyr; t-butyl ester Glu and Asp Trityl Cys, His,Asn, and Gln 2,2,5,7,8-pentamethylchromane-6- Arg sulfonylbutoxycarbonyl (tBoc) Lys

The carboxylate group of the incoming Fmoc-protected amino acid isactivated in order to achieve efficient chemical coupling to theN-terminus of the resin-bound polypeptide. Activation is typicallyaccomplished by reacting an Fmoc-protected amino acid with a suitablereagent to yield a reactive ester. Examples of activated esters includethe pentafluorophenyl (OPfp) ester and the3-hydroxy-2,3-dihydro-4-oxo-benzo-triazone (ODhbt) ester, OBt ester, andthe OAt ester derived from 1-hydroxy-7-azabenzotriazole (HOAt). Thecoupling reactions can be done in situ using activating reagents, suchas DCC, BOP, BOP-Cl, TBTU, HBTU or O-(7-azabenzotrizol-1-yl)-1,1,3,3,tetramethyluronium hexafluorophosphate (HATU). Exemplary couplingreactions included a mixture of HOBt and HBTU, or a mixture of HOBt,HBTU, and DIEA. For N-methyl amino acids, coupling conditions can usebromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBroP) as theonly coupling reagent, and the coupling reaction is performed manuallyin DCM with DIEA present under N₂. The Fmoc-protected amino acid ispresent in molar excess to the polypeptide coupled to the resin. Forcoupling reactions that proceed with a slow rate, the coupling reactionsare repeated one or more times (double or multiple coupling) to ensurethat all resin-bound polypeptide has undergone a successful additionreaction with the activated Fmoc-amino acid. For incomplete couplingreactions, any un-reacted N-terminal residues are capped using asuitable capping reagent.

Following the coupling reaction, the resin support is washed to removethe unreacted Fmoc-amino acids and coupling reagents. The resin is thensubjected to a new cycle of base-catalyzed removal of the N-terminalFmoc group to prepare the polypeptide for another amino acid addition.After the desired polypeptide has been synthesized, the resin issubjected to base-catalyzed removal of the remaining Fmoc protectiongroup. The polypeptide-coupled resin is washed to remove the base andsubsequently treated with acid to remove any amino acid side chainprotecting groups and to release the polypeptide chain from the resinsupport. Useful acids are strong acids, such as trifluoroacetic acid(TFA) in the presence of suitable scavengers, such as reagent K[TFA:thioanisole:ethanedithiol:phenol:water (82.5:5:2.5:5:5)].

The polypeptide is subsequently separated from the resin by filtrationand optionally washed repeatedly with a suitable solvent, such asDCM/DMF. The polypeptide can be optionally desalted throughultrafiltration using a membrane with a suitable MW cutoff. Thepolypeptide can be precipitated from solution using a suitable solvent,such as cold methyl t-butyl ether or t-butylethylether, and theprecipitate optionally washed with a suitable solvent, such as coldether and dried. The polypeptide can be further purified using asuitable chromatographic means, such as hydrophobic chromatography usinga C18 resin and an appropriate chromatographic buffer system, such asTFA/water/acetonitrile. The purity of the peptide optionally can beanalyzed by mass spectrometry, such as MALDI-MS, analytical HPLC, aminoacid analysis or sequencing.

Alternatively, the TCAs of SEQ ID NOs.:2, 4, 6, and 8 can be expressedrecombinantly using the polynucleotide sequences of SEQ ID NOs.:1, 3, 5and 7 using, for example, expression vectors. In expression vectors, theintroduced DNA is operably-linked to elements, such as promoters, thatsignal to the host cell to transcribe the inserted DNA. Some promotersare exceptionally useful, such as inducible promoters that control genetranscription in response to specific factors. Examples of induciblepromoters include those that are tissue-specific, which relegateexpression to certain cell types, steroid-responsive (e.g.,glucocorticoids (Kaufman, R. J. 1990. Vectors used for expression inmammalian cells. Methods Enzymol. 185:487-511) and tetracycline, orheat-shock reactive. Some bacterial repression systems, such as the lacoperon, can be exploited in mammalian cells and transgenic animals(Fieck, A., et al. 1992. Modifications of the E. coli lac repressor forexpression in eukaryotic cells: effects of nuclear signal sequences onprotein activity and nuclear accumulation. Nucleic Acids Res.20:1785-91; Wyborski, D. L., L. C. DuCoeur, and J. M. Short. 1996).Parameters affecting the use of the lac repressor system in eukaryoticcells and transgenic animals. Environ Mol Mutagen. 28:447-58; Wyborski,D. L., and J. M. Short. 1991. Analysis of inducers of the E. coli lacrepressor system in mammalian cells and whole animals. Nucleic AcidsRes. 19:4647-53). Recombinant nucleic acid technologies, transfectioninto cells and cellular and in vitro expression are discussed furtherbelow.

E. Recombinant Antibodies

The recombinant antibodies of the present disclosure compriseantigen-binding regions derived from the V_(H) and/or V_(L) domains of anative antibody capable of specifically binding to a T. cruzi antigenicprotein. The recombinant antibody can be, for example, arecombinantly-produced monoclonal antibody, a fragment of a monoclonalantibody, a chimeric antibody, a humanized antibody, a multispecific,dual-variable domain immunoglobulins (DVD-Ig®) or multivalent structureformed from an antibody fragment, or a bifunctional antibody.

In one embodiment, optionally, the recombinant antibody is an antibodythat: (a) that specifically binds to a diagnostically relevant region ofa T. cruzi polypeptide, wherein the T. cruzi polypeptide is FRA andfurther wherein said antibody has at last one binding constant selectedfrom the group consisting of: an association rate constant (k_(a))between about 7.0×10⁵M⁻¹ s⁻¹ to about 7.0×10⁶M⁻¹ s⁻¹, an dissociationrate constant (k_(d)) between about 4.0×10⁻³ s⁻¹ to about 3.0×10⁻¹ s⁻¹and an equilibrium dissociation constant (K_(D)) between about 5.7×10⁻¹⁰M to about 4.3×10⁻⁷ M;

(b) that specifically binds to a diagnostically relevant region of a T.cruzi polypeptide, wherein the T. cruzi polypeptide is Pep2 and furtherwherein said antibody has at least one binding constant selected fromthe group consisting of: an association rate constant (k_(a)) betweenabout 1.0×10⁶M⁻¹ s⁻¹ to about 8.0×10⁶M⁻¹ s⁻¹; an dissociation rateconstant (k_(d)) between about 6.0×10⁻³ s⁻¹ to about 4.0×10⁻² s¹ and anequilibrium dissociation constant (K_(D)) between about 7.5×10⁻¹⁰ M toabout 4.0×10⁻⁸ M;

(c) that specifically binds to a diagnostically relevant region of a T.cruzi polypeptide, wherein the T. cruzi polypeptide is FP10 and furtherwherein said antibody has at least one binding constant selected fromthe group consisting of: (a) an association rate constant (k_(a))between about 5.0×10⁴M⁻¹ s⁻¹ to about 3.0×10⁵M⁻¹ s⁻¹: (b) andissociation rate constant (k_(d)) between about 1.0×10⁻⁴ s⁻¹ to about8.0×10⁻⁴ s⁻¹; and (c) an equilibrium dissociation constant (K_(D))between about 3.3×10⁻¹⁰ M to about 1.6×10⁻⁸M;

(d) that specifically binds to a diagnostically relevant region of a T.cruzi polypeptide, wherein the T. cruzi polypeptide is FP3 and furtherwherein said antibody has at least one binding constant selected fromthe group consisting of: an association rate constant (k_(a)) betweenabout 2.0×10⁵M⁻¹ s⁻¹ to about 6.0×10⁶M⁻¹ s⁻¹; an dissociation rateconstant (k_(d)) between about 2.0×10⁻⁵ s⁻¹ to about 8.0×10⁻⁴ s⁻¹; andan equilibrium dissociation constant (K_(D)) between about 3.3×10⁻¹²M toabout 4.0×10⁻⁹ M; and

(e) any combinations of (a)-(d). In another embodiment, optionally, therecombinant antibody is a chimeric antibody that retains the mousemonoclonal antibody specificity and affinity and reacts in animmunoassay format that measures human immunoglobulin. Optionally, themouse-human chimeric antibody is directed against the FP3, FP6, FP10 orFRA antigen. Optionally, such a chimeric antibody reacts in an existingimmunoassay format including, but not limited to, Abbott Laboratories'AxSYM®, ARCHITECT® and PRISM® platforms.

The antigen-binding region comprised by the recombinant antibody caninclude the entire V_(H) and/or V_(L) sequence from the native antibody,or it can comprise one or more portions thereof, such as the CDRs,together with sequences derived from one or more other antibodies. Inone embodiment, the recombinant antibody comprises the full-length V_(H)and V_(L) sequences of the native antibody.

The native antibody from which the antigen-binding regions are derivedis generally a vertebrate antibody. For example, the native antibody canbe a rodent (e.g., mouse, hamster, rat) antibody, a chicken antibody, arabbit antibody, a canine antibody, a feline antibody, a bovineantibody, an equine antibody, a porcine antibody, an ape (e.g.,chimpanzee) antibody, or a human antibody. The source of the antibody isbased primarily on convenience. In one embodiment, the native antibodyis a non-human antibody.

The recombinant antibody also can include one or more constant regions,for example, the C_(L), C_(H)l hinge, C_(H)2, C_(H)3, and/or C_(H)4regions, derived from the same native antibody or from a differentantibody. The constant region(s) can be derived from an antibody fromone of a number of vertebrate species, including but not limited to,those listed above. In one embodiment, of the present disclosure, therecombinant antibody comprises at least one constant region. In anotherembodiment, the recombinant antibody comprises one or more constantregions that are derived from a human antibody. In a specific embodimentof the present disclosure, the recombinant antibody comprises thevariable region of a non-human antibody linked to the constant region ofa human antibody.

The constant region(s) comprised the recombinant antibody can be derivedfrom one or more immunoglobulin classes or isotypes, for example forconstant regions derived from human immunoglobulins, the constant regioncan be derived from one or more of an IgM, IgD, IgG1, IgG2, IgG3, IgG4,IgA1, IgA2 or IgE constant region. When the constant region comprises aregion derived from an IgG light chain, this can be derived from a kappachain or a lambda chain. The recombinant antibody can comprise sequencesfrom more than one class or isotype. Selection of particular constantdomains to optimize the desired function of the recombinant antibody iswithin the ordinary skill in the art. In one embodiment, of the presentdisclosure, the recombinant antibody comprises one or more constantdomains derived from an IgG. In another embodiment, the recombinantantibody comprises regions from both the heavy and light chains of anIgG constant domain.

In one embodiment, of the present disclosure, the antigen-bindingregions are derived from a native antibody that specifically binds to anepitope within a diagnostically relevant region of a T. cruzi antigenicprotein.

In a specific embodiment of the present disclosure, the antigen-bindingregions of the recombinant antibody comprise an amino acid sequencesubstantially identical to all or a portion of the V_(H) or V_(L)sequence as set forth in any one of SEQ ID NOs.:10, 12, 14, 16, 18, 20,26 or 28 (See, Table 12 below; See, Table 11 below for a summary of SEQID NO identifiers and the corresponding sequence descriptions). Inanother embodiment, the antigen-binding regions of the recombinantantibody comprise the complementarity determining regions (CDRs; i.e.,CDR1, CDR2 and CDR3) of a V_(H) or V_(L) sequence.

TABLE 11 Summary of SEQ ID NOs.: for V_(L) and V_(H) chains V_(L) V_(L)V_(H) V_(H) Poly- Poly- Poly- Poly- Antigen Cell line nucleotide peptidenucleotide peptide FP3 HBFP3 9 10 11 12 FP6 HBPep2 13 14 15 16(TcF/Pep2) FP10 HBFP10 17 18 19 20 FRA 8-367-171 25 26 27 28

TABLE 12 Exemplary V_(H) and V_(L) Polypeptide Sequences SEQ ID V_(H) orNO.: Sequence V_(L) TCA 10 YIVMSQSPSS LAVSAGEKVT MSCKSSQSLL  V_(L) FP3NSRTRKNHLA WYQQKPGQSP KLLIYWASTR ESGVPDRFTG SGSGTDFALT ISSVQAEDLAVYFCKQSYNL YTFGAGTKLE LK 12 DVQLVESGGG LVQPGGSRKL SCAASGFTFS  V_(H) FP3VFGMHWVRQA PEKGLEWVAY ISSGSTIIYY ADTVKGRFTI SRDNPKNTLF LQMTGLRSEDTAMYYCARPL YYDYDDYAMD YWGQGTSVTV SS 14 DIVMSQSPSS LAVSAGEQVT MSCKSSQSLF V_(L) FP6 NSRTRKNYLA WYQQKPGQSP KLLIYWASTRESGVPDRFTG SGSGTDFILT ISSVQAEDLA VYYCKQSYNL LTFGAGTKLE LK 16QVQLQQPGAE LVRPGASVKL SCKASGYTFT  V_(H) FP6SYWMNWVKLR PGQGLEWIGM IDPSDSETYY DQVFKDKATL TVDKSSSTAY MHLSSLTSEDSAVYYCARWI TTDSYTMDYW GQGTSVTVSS 18 DVVMTQTPLS LPVSLGDQAS ISCRSSQSLV V_(L) FP10 HSNGNTYLHW YLQKPGQSPK LLIYKVSNRFSGVPDRFSGS GSGTDFTLKI SRVEAEDLGV YFCSQSTHVP PTFGGGTKLE IK 20QVQLQQPGAE LVKPGASVKM SCKASGYTFT  V_(H) FP10SYWVHWVKQR PGQGLEWIGV IDPSDSYTSY NQKFKGKATL TVDTSSSTAY MQLSSLTSEDSAVYYCTRHY DFDSWYFDVW GAGTTVTVSS 26 DIQMDQSPSS LSASLGDTIT ITCHASQNIN V_(L) FRA VWLSWYQQKP GNIPKLLIYK ASNLHTGVPSRFSGSGSGTG FTLTISSLQP EDIATYYCQQ GQSYPLITGS GRKLEIK 28EVQLQQSGAE LVKPGASVKL SCTASGFNIK  V_(H) FRADTYMHWVKQR PEQGLEWIGR IDPANGNTKY DPKFQGKATI TTDTSSNTAY LQLSSLTSEDTAVYYCATSY YGNYVAYWGH GTLVTVSA

In one embodiment, of the present disclosure, the antigen-bindingregions of the recombinant antibody comprise an amino acid sequencesubstantially identical to all or a portion of the amino acid sequenceencoded by any one of SEQ ID NOs.:9, 11, 13, 15, 17, 19, 25 or 27 (See,Table 13, below). In another embodiment, the antigen-binding regions ofthe recombinant antibody comprise a nucleic acid sequence encoding thecomplementarity determining regions (CDRs; i.e., CDR1, CDR2 and CDR3) ofa V_(H) or V_(L) sequence. In a specific embodiment, the antigen-bindingregions of the recombinant antibody comprise CDRs having an amino acidsequence substantially identical to the amino acid sequences encoded byone or more of SEQ ID NOs.:9 and 11; one or more of SEQ ID NOs.:13 and15; or one or more of SEQ ID NOs.:17 and 19; or one or more of SEQ IDNOs.:25 or 27 (See, Table 13, below).

In another specific embodiment of the present disclosure, theantigen-binding regions of the recombinant antibody comprise an aminoacid sequence encoded by a nucleic acid sequence substantially identicalto all or a portion of the sequence as set forth in any one of SEQ IDNOs.:9, 11, 13, 15, 17, 19, 25 or 27.

TABLE 13 Exemplary Nucleic Acid Sequences EncodingV_(H) and V_(L) Sequences SEQ ID V_(H) or NO.: Sequence V_(L) TCA  9TACATTGTGA TGTCACAGTC TCCATCCTCC  V_(L) FP3CTGGCTGTGT CAGCAGGAGA GAAGGTCACT ATGAGCTGCA AATCCAGTCA GAGTCTGCTCAACAGTAGAA CCCGAAAGAA CCACTTGGCT TGGTATCAGC AGAAACCAGG GCAGTCTCCT AAACTGCTGA TCTACTGGGC ATCCACTAGG GAATCTGGGG TCCCTGATCG CTTCACAGGCAGTGGATCTG GGACAGATTT CGCTCTCACC ATCAGCAGTG TGCAGGCTGA AGACCTGGCA GTTTATTTCT GCAAGCAATC TTATAATCTG TACACATTCG GTGCTGGGAC CAAGCTGGAG CTGAAA11 GATGTGCAGC TGGTGGAGTC TGGGGGAGGC  V_(H) FP3TTAGTGCAGC CTGGAGGGTC CCGGAAACTC TCCTGTGCAG CCTCTGGATT CACTTTCAGTGTCTTTGGAA TGCACTGGGT TCGTCAGGCT CCAGAGAAGG GGCTGGAGTG GGTCGCATAC ATTAGTAGTG GCAGTACTAT CATCTATTAT GCAGACACAG TGAAGGGCCG ATTCACCATCTCCAGAGACA ATCCCAAGAA CACCCTGTTC CTGCAAATGA CCGGTCTAAG GTCTGAGGAC ACGGCCATGT ATTACTGTGC AAGACCGCTC TACTATGATT ACGACGACTA TGCTATGGACTACTGGGGTC AAGGAACCTC AGTCACCGTC TCCTCA 13GACATTGTGA TGTCACAGTC TCCATCCTCC  V_(L) FP6CTGGCTGTGT CAGCAGGAGA GCAGGTCACT ATGAGCTGCA AATCCAGTCA GAGTCTGTTCAACAGTAGAA CCCGAAAGAA CTACTTGGCT TGGTACCAGC AGAAACCAGG GCAGTCTCCT AAACTGCTGA TCTACTGGGC ATCCACTAGG GAATCTGGGG TCCCTGATCG CTTCACAGGCAGTGGATCTG GGACAGATTT CACTCTCACC ATCAGCAGTG TGCAGGCTGA AGACCTGGCA GTTTATTACT GCAAACAATC TTATAATCTG CTCACGTTCG GTGCTGGGAC CAAGCTGGAG CTGAAA15 CAGGTCCAAC TGCAGCAGCC TGGGGCTGAA  V_(H) FP6 CTGGTGAGGC CTGGGGCTTCAGTGAAACTGTCCTGCAAGG CTTCTGGCTA CACCTTCACC AGCTACTGGA TGAACTGGGT GAAGTTGAGG CCTGGACAAG GCCTTGAATG GATTGGTATG ATTGATCCTT CAGACAGTGAAACTTACTAC GATCAAGTAT TCAAGGACAA GGCCACATTG ACTGTTGACA AATCCTCCAG CACAGCCTAC ATGCATCTCA GCAGCCTGAC ATCTGAGGAC TCTGCGGTCT ATTACTGTGCAAGATGGATT ACGACTGATT CCTATACTAT GGACTACTGG GGTCAAGGAA CCTCAGTCAC CGTCTCCTCA 17 GATGTTGTGA TGACCCAAAC TCCACTCTCC  V_(L) FP10CTGCCTGTCA GTCTTGGAGA TCAAGCCTCC ATCTCTTGCA GATCTAGTCA GAGCCTTGTACACAGTAATG GAAACCCTAT TTACATTGGT ACCTGCAGAA GCCAGGCCAG TCTCCAAAGC TCCTGATCTA CAAAGTTTCC AACCGATTTT CTGGGGTCCC AGACAGGTTC AGTGGCAGTGGATCAGGGAC AGATTTCACA CTCAAGATCA GCAGAGTGGA GGCTGAGGAT CTGGGAGTTT ATTTCTGCTC TCAAAGTACA CATGTTCCTC CGACGTTCGG TGGAGGCACC AAGCTGGAAA TCAAA19 CAGGTCCAAC TGCAGCAGCC TGGGGCTGAG  V_(H) FP10CTGGTGAAGC CTGGGGCTTC AGTGAAGATG TCCTGCAAGG CTTCTGGCTA CACCTTCACCAGCTACTGGG TGCACTGGGT GAAGCAGAGG CCTGGACAAG GCCTTGAGTG GATCGGAGTG ATTGATCCTT CTGATAGTTA TACTAGCTAC AATCAAAAGT TCAAGGGCAA GGCCACATTACTGTAGACAC ATCCTCCAGC ACAGCCTACA TGCAGCTCAG CAGCCTGACA TCTGAGGACT CTGCGGTCTA TTACTGTACA AGACACTATG ATTTCGACAG CTGGTACTTC GATGTCTGGGGCGCAGGGAC CACGGTCACC GTCTCCTCA 25 gacatccaga tggaccagtc tccatccagt V_(L) FRA ctgtctgcat cccttggaga cacaattaccatcacttgcc atgccagtca gaacattaat gtttggttaa gctggtacca gcagaaaccaggaaatattc ctaaactatt gatctataag  gcttccaact tgcacacagg cgtcccatcaaggtttagtg gcagtggatc tggaacaggt ttcacattaa ccatcagcag cctgcagcctgaagacattg ccacttacta ctgtcaacag  ggtcaaagtt atcctctcac gttcggctcggggcgaaagt tggaaataaa a 27 gaggttcagc tgcagcagtc tggggcagag  V_(H) FRActtgtgaagc caggggcctc agtcaagttg tcctgcacag cttctggctt caacattaaagacacctata tgcactgggt gaagcagagg cctgaacagg gcctggagtg gattggaagg attgatcctg cgaatggtaa tactaaatat gacccgaagt tccagggcaa ggccactataacaacagaca catcctccaa cacagcctac ctgcagctca gcagcctgac atctgaggac actgccgtct attactgtgc tacctcctac tatggtaact acgttgctta ctggggccacgggactctgg tcactgtctc tgca

The amino acid sequence of recombinant antibody need not correspondprecisely to the parental sequences, i.e., it can be a “variantsequence.” For example, depending in the domains comprised by therecombinant antibody, one or more of the V_(L), V_(H), C_(L), C_(H)1,hinge, C_(H)2, C_(H)3, and C_(H)4, as applicable, can be mutagenized bysubstitution, insertion or deletion of one or more amino acid residuesso that the residue at that site does not correspond to either theparental (or reference) sequence. One skilled in the art willappreciate, however, that such mutations will not be extensive and willnot significantly affect binding of the recombinant antibody to itstarget TCA. In accordance with the present disclosure, when arecombinant antibody comprises a variant sequence, the variant sequenceis at least about 70% (e.g., from about 70% to about 100%) identical tothe reference sequence. In one embodiment, the variant sequence is atleast about 75% (e.g., from about 75% to about 100%) identical to thereference sequence. In other embodiments, the variant sequence is atleast about 80% (e.g., from about 80% to about 100%), at least about 85%(e.g., from about 85% to about 100%), or at least about 90% (e.g., fromabout 90% to about 100%) identical to the reference sequence. In aspecific embodiment, the reference sequence corresponds to a sequence asset forth in any one of SEQ ID NOs.:10, 12, 14, 16, 18, 20, 26 or 28.

Generally, when the recombinant antibody comprises a variant sequencethat contains one or more amino acid substitutions, they are“conservative” substitutions. A conservative substitution involves thereplacement of one amino acid residue by another residue having similarside chain properties. As is known in the art, the twenty naturallyoccurring amino acids can be grouped according to the physicochemicalproperties of their side chains. Suitable groupings include alanine,valine, leucine, isoleucine, proline, methionine, phenylalanine andtryptophan (hydrophobic side chains); glycine, serine, threonine,cysteine, tyrosine, asparagine, and glutamine (polar, uncharged sidechains); aspartic acid and glutamic acid (acidic side chains) andlysine, arginine and histidine (basic side chains). Another grouping ofamino acids is phenylalanine, tryptophan, and tyrosine (aromatic sidechains). A conservative substitution involves the substitution of anamino acid with another amino acid from the same group.

Thus, the present disclosure in other embodiments further providesisolated polypeptides corresponding to novel recombinant antibodysequences disclosed herein. Optionally the isolated polypeptidecomprises a portion of a recombinant (e.g., chimeric) antibody thatspecifically binds to a diagnostically relevant region of a TCA selectedfrom the group consisting of FP3, FP6, and FP10. In one embodiment, thepolypeptide comprises a V_(H) region selected from the group consistingof a V_(H) region comprising an amino acid sequence substantiallyidentical to the sequence as set forth in any one or more of SEQ IDNOs.:12, 16, 20 or 28. In still another embodiment, the polypeptidecomprises a V_(H) region comprising complementarity determining regionsequences. In another embodiment, the polypeptide comprises a V_(L)region comprising an amino acid sequence that is substantially identicalto the sequence as set forth in any one or more of SEQ ID NOS.:10, 14,18 or 26. In still another embodiment, the polypeptide comprises a V_(L)region comprising complementarity determining region sequences.

In still another embodiment, the polypeptide comprises a V_(H) regionselected from the group consisting of a V_(H) region comprising an aminoacid sequence substantially identical to the sequence encoded by any oneor more of SEQ ID NOs.:11, 15, 19 or 27. In another embodiment, thepolypeptide comprises a V_(L) region selected from the group consistingof a V_(L) region comprising an amino acid sequence substantiallyidentical to the sequence encoded by any one or more of SEQ ID NOs.:9,13, 17 or 25.

Likewise, the nucleic acid sequence encoding the variable region(s) neednot correspond precisely to the parental reference sequence but can varyby virtue of the degeneracy of the genetic code and/or such that itencodes a variant amino acid sequence as described above. In oneembodiment, of the present disclosure, therefore, the nucleic acidsequence encoding a variable region of the recombinant antibody is atleast about 70% (e.g., from about 70% to about 100%) identical to thereference sequence. In another embodiment, the nucleic acid sequenceencoding a variable region of the recombinant antibody is at least about75% (e.g., from about 75% to about 100%) identical to the referencesequence. In other embodiments, the nucleic acid sequence encoding avariable region of the recombinant antibody is at least about 80% (e.g.,from about 80% to about 100%), at least about 85% (e.g., from about 85%to about 100%), or at least about 90% (e.g., from about 90% to about100%) identical to the reference sequence. In a specific embodiment, thereference sequence corresponds to a sequence as set forth in any one ofSEQ ID NOs.:9, 11, 13, 15, 17, 19 25 or 27.

Thus, the present disclosure in other embodiments further providesisolated polynucleotides which encode novel recombinant antibodysequences, including chimerical antibody sequences, disclosed herein.Optionally, the isolated polynucleotide encodes a portion of arecombinant (e.g., chimeric) antibody that specifically binds to adiagnostically relevant region of a T. cruzi protein selected from thegroup consisting of FP3, FP6 and FP10 protein. In one embodiment, thepolynucleotide encodes a V_(H) region selected from the group consistingof a V_(H) region comprising an amino acid sequence substantiallyidentical to the sequence as set forth in any one or more of SEQ IDNOs.:12, 16, 20 or 28. In another embodiment the polynucleotide encodesa V_(L) region comprising an amino acid sequence that is substantiallyidentical to the sequence as set forth in any one or more of SEQ IDNOs.:10, 14, 18 or 26. In still another embodiment, the polynucleotideencodes a V_(L) region comprising complementarity determining regionsequences.

In still another embodiment, the polynucleotide encodes a V_(H) regionselected from the group consisting of a V_(H) region comprising an aminoacid sequence substantially identical to the sequence encoded by any oneor more of SEQ ID NOs.:9, 13, 17 or 27. In yet another embodiment, thepolynucleotide encodes a V_(H) region comprising complementaritydetermining region sequences. In another embodiment, the polynucleotideencodes a V_(L) region selected from the group consisting of a V_(L)region comprising an amino acid sequence substantially identical to thesequence encoded by any one or more of SEQ ID NOs.:11, 15, 19 or 25. Instill yet another embodiment, the polynucleotide encodes a V_(L) regioncomprising complementarity determining region sequences.

In one embodiment, the antibodies can be further modified to reduce theimmunogenicity to a human relative to the native antibody by mutatingone or more amino acids in the non-human portion of the antibody thatare potential epitopes for human T-cells in order to eliminate or reducethe immunogenicity of the antibody when exposed to the human immunesystem. Suitable mutations include, for example, substitutions,deletions and insertions of one or more amino acids.

In one embodiment, the recombinant antibodies of the present disclosurecan be further modified for immobilization onto a suitable solid phaseImmobilization can be achieved through covalent or non-covalent (forexample, ionic, hydrophobic, or the like) attachment to the solid phase.Suitable modifications are known in the art and include the addition ofa functional group or chemical moiety to either the C-terminus or theN-terminus of one of the amino acid sequences comprised by therecombinant antibody to facilitate cross-linking or attachment of therecombinant antibody to the solid support. Exemplary modificationsinclude the addition of functional groups such asS-acetylmercaptosuccinic anhydride (SAMSA) or S-acetyl thioacetate(SATA), or addition of one or more cysteine residues to the N- orC-terminus of the amino acid sequence. Other cross-linking reagents areknown in the art, and many are commercially available (see, for example,catalogues from Pierce Chemical Co. (Rockford, Ill., USA) andSigma-Aldrich; Saint Louis, Mo., USA). Examples include, but are notlimited to, diamines, such as 1,6-diaminohexane; dialdehydes, such asglutaraldehyde; bis-N-hydroxysuccinimide esters, such as ethyleneglycol-bis(succinic acid N-hydroxysuccinimide ester), disuccinimidylglutarate, disuccinimidyl suberate, and ethyleneglycol-bis(succinimidylsuccinate); diisocyantes, such ashexamethylenediisocyanate; bis oxiranes, such as 1,4 butanediyldiglycidyl ether; dicarboxylic acids, such as succinyidisalicylate;3-maleimidopropionic acid N-hydroxysuccinimide ester, and the like.

Other modifications include the addition of one or more amino acids atthe N- or C-terminus, such as histidine residues to allow binding toNi²⁺ derivatized surfaces, or cysteine residues to allow disulfidebridge formation or binding to SULFOLIIK™ agarose. Alternatively, theantibody can be modified to include one or more chemical spacers at theN-terminus or C-terminus in order to distance the recombinant antibodyoptimally from the solid support. Spacers that can be used include, butare not limited to, 6-aminohexanoic acid; 1,3-diamino propane;1,3-diamino ethane; and short amino acid sequences, such as polyglycinesequences, of 1 to 5 amino acids.

In an alternative embodiment, the recombinant antibodies optionally canbe conjugated to a carrier protein, such as bovine serum albumin (BSA),casein, or thyroglobulin, in order to immobilize them onto a solidphase.

In another embodiment, the present disclosure provides for modificationof the recombinant antibodies to incorporate a detectable label.Detectable labels according to the disclosure preferably are moleculesor moieties which can be detected directly or indirectly and are chosensuch that conjugation of the detectable label to the recombinantantibody preferably does not interfere with the specific binding of theantibody to its target T. cruzi protein. Methods of labeling antibodiesare well-known in the art and include, for example, the use ofbifunctional cross-linkers, such as SAMSA (S-acetylmercaptosuccinicanhydride), to link the recombinant antibody to the detectable label.Other cross-linking reagents such as are known in the art or whichsimilar to those described above likewise can be used.

Detectable labels for use with the recombinant antibodies of the presentdisclosure include, for example, those that can be directly detected,such as radioisotopes, fluorophores, chemiluminophores, enzymes,colloidal particles, fluorescent microparticles, and the like. Thedetectable label is either itself detectable or can be reacted with oneor more additional compounds to generate a detectable product. Thus, oneskilled in the art will understand that directly detectable labels ofthe disclosure can require additional components, such as substrates,triggering reagents, light and the like to enable detection of thelabel. Examples of detectable labels include, but are not limited to,chromogens, radioisotopes (such as, e.g., ¹²⁵I, ¹³¹I, ³²P, ³H, ³⁵S and¹⁴C), fluorescent compounds (such as fluorescein, rhodamine, rutheniumtris bipyridyl and lanthanide chelate derivatives), chemiluminescentcompounds (such as, e.g., acridinium and luminol), visible orfluorescent particles, nucleic acids, complexing agents, or catalystssuch as enzymes (such as, e.g., alkaline phosphatase, acid phosphatase,horseradish peroxidase, β-galactosidase, β-lactamase, luciferase). Inthe case of enzyme use, addition of, for example, a chromo-, fluoro-, orlumogenic substrate preferably results in generation of a detectablesignal. Other detection systems such as time-resolved fluorescence,internal-reflection fluorescence, and Raman spectroscopy are optionallyalso useful.

The present disclosure also provides for the use of labels that aredetected indirectly. Indirectly detectable labels typically involve theuse of an “affinity pair,” i.e., two different molecules, where a firstmember of the pair is coupled to the recombinant antibody of the presentdisclosure, and the second member of the pair specifically binds to thefirst member. Binding between the two members of the pair is typicallychemical or physical in nature. Examples of such binding pairs include,but are not limited to: antigens and antibodies; avidin/streptavidin andbiotin; haptens and antibodies specific for haptens; complementarynucleotide sequences; enzyme cofactors/substrates and enzymes; and thelike.

F. Preparation of Antibodies

Polyclonal Abs can be raised in a mammalian host by one or moreinjections of an immunogen and, if desired, an adjuvant. Typically, theimmunogen (and adjuvant) is injected in the mammal by multiplesubcutaneous or intraperitoneal injections. The immunogen can include aTCA or a TCA-fusion polypeptide. Examples of adjuvants include Freund'scomplete and monophosphoryl Lipid A synthetic-trehalose dicorynomycolate(MPL-TDM). To improve the immune response, an immunogen can beconjugated to a polypeptide that is immunogenic in the host, such askeyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin,and soybean trypsin inhibitor. Protocols for antibody production arewell-known (Ausubel et al., 1987; Harlow, E., and D. Lane. 1988.Antibodies: A laboratory manual. Cold Spring Harbor Laboratory Press,Cold Spring Harbor. 726 pp; Harlow, E., and D. Lane. 1999. Usingantibodies: A laboratory manual. Cold Spring Harbor Laboratory PRess,Cold Spring Harbor, N.Y.). Alternatively, pAbs can be made in chickens,producing IgY molecules (Schade, R., et al. 1996. The production ofavian (egg yolk) antibodies: IgY. The report and recommendations ofECVAM workshop. Alternatives to Laboratory Animals (ATLA). 24:925-934).

Methods of raising monoclonal antibodies against a desired antigen arewell known in the art. For example, monoclonal antibodies can be madeusing the hybridoma method first described by Kohler et al., Nature,256:495 (1975). In general in the hybridoma method, a mouse or otherappropriate host animal, such as a hamster or macaque monkey, isimmunized by multiple subcutaneous or intraperitoneal injections ofantigen and a carrier and/or adjuvant at multiple sites. Two weekslater, the animals are boosted, and about 7 to 14 days later animals arebled and the serum is assayed for anti-antigen titer. Animals can beboosted until titer plateaus.

The splenocytes of the mice are extracted and fused with myeloma cellsusing a suitable fusing agent, such as polyethylene glycol, to form ahybridoma cell (see, for example, Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986); Galfre etal., Nature, 266:550 (1977)). Suitable myeloma cell lines are known inthe art and include, but are not limited to, murine myeloma lines, suchas those derived from MOP-21 and MC-11 mouse tumors (available from theSalk Institute Cell Distribution Center, San Diego, Calif., USA), aswell as SP-2, SP2/0 and X63-Ag8-653 cells (available from the AmericanType Culture Collection (ATCC), Manassas, Va., USA). Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (see, for example, Kozbor, J.Immunol., 133:3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987)). The hybridoma cells thus prepared can be seeded andgrown in a suitable culture medium that preferably contains one or moresubstances that inhibit the growth or survival of the unfused, parentalmyeloma cells. For example, if the parental myeloma cells lack theenzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT),the culture medium for the hybridomas typically will includehypoxanthine, aminopterin, and thymidine (HAT medium), which substancesprevent the growth of HGPRT-deficient cells.

The hybridoma cells obtained through such a selection are then assayedto identify clones which secrete antibodies capable of binding the T.cruzi antigen used in the initial immunization, for example, byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-immunoassay (EIA or ELISA). The bindingaffinity of the monoclonal antibody can optionally be determined, forexample, by the Scatchard analysis of Munson et al., Anal. Biochem.,107:220 (1980). After hybridoma cells are identified that produceantibodies of the desired specificity, the clones can be subcloned bylimiting dilution procedures, for example the procedure described byWands et al. (Gastroenterology 80:225-232 (1981)), and grown by standardmethods (see, for example, Goding, ibid.). Suitable culture media forthis purpose include, for example, D-MEM, IMDM or RPMI-1640 medium.Alternatively, the hybridoma cells can be grown in vivo as ascitestumors in an animal.

The monoclonal antibodies secreted by the subclones optionally can beisolated from the culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A chromatography, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

Examples 1-4 (See, the Example section) illustrate just one approach toobtaining mAbs to the TCAs found in FP3, FP6, FP10 and FRA polypeptides(e.g., the polypeptides represented by the amino acid sequences of SEQID NOs.:2, 4, 6 and 8).

G. Preparation of Recombinant Antibodies

The recombinant antibodies of the present disclosure can compriseantigen-binding domain sequences (for example, the V_(H) and/or V_(L)sequences, or a portion thereof) derived from, for example, a monoclonalantibody produced by a human or non-human animal, such as a rodent,rabbit, canine, feline, bovine, equine, porcine, ape or chicken.Alternatively, antigen-binding domains with the desired binding activitycan be selected through the use of combinatorial libraries expressed inlambda phage, on the surface of bacteriophage, bacteria or yeast, orscreened by display on other biological (for example, retrovirus orpolysome) or non-biological systems using standard techniques (See, forexample, Marks, J. D. et. al., J. Mol. Biol. 222:581-597 (1991); Barbas,C. F. III et. al., Proc. Natl. Acad. Sci. USA 89:4457-4461 (1992)). Thelibraries can be composed of native antigen-binding domains isolatedfrom an immunized or unimmunized host, synthetic or semi-syntheticantigen-binding domains, or modified antigen-binding domains.

1. Recombinant Abs Generally

In one embodiment of the present disclosure, the recombinant antibodiescomprise antigen-binding domains derived from monoclonal antibodies thatbind to the T. cruzi protein of interest.

In one embodiment of the present disclosure, the recombinant antibodiesare derived from monoclonal antibodies raised to a T. cruzi antigenderived from a diagnostically relevant region of a T. cruzi protein. Inanother embodiment, the recombinant antibodies are derived frommonoclonal antibodies raised to a T. cruzi antigen, such as FP3, FP6, orFP10. In another embodiment, the recombinant antibodies are derived frommonoclonal antibodies raised to a T. cruzi antigen comprising all or afragment (for example, a fragment comprising one or more epitopes) ofFP3, FP6 or FP10. In a further embodiment, the recombinant antibodiesare derived from monoclonal antibodies raised to a T. cruzi antigencomprising a sequence substantially identical to the sequence as setforth in any one of SEQ ID NOs.:2, 4 or 6.

Optionally, the monoclonal antibody is expressed by a cell line selectedfrom the group consisting of HBFP3, HBPep2, and HBFP10. In analternative embodiment, the cell line is Chagas 8-367-171.

Once a monoclonal antibody has been prepared, DNA encoding themonoclonal antibody or the variable regions thereof can readily beisolated by standard techniques, for example by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains or the variable regions of the monoclonalantibody, or by RT-PCR of the mRNA encoding the monoclonal antibodyusing primers to conserved regions (for example, the IgG primer setsavailable from Novagen (EMD Biosciences, Inc.), San Diego, Calif., USA).

Once isolated, the DNA can be, for example, cloned into an appropriateexpression vector and introduced into a suitable host cell, such as E.coli cells, yeast cells, simian COS cells, Chinese hamster ovary (CHO)cells, human embryonic kidney (HEK) cells (for example, HEK 293), ormyeloma cells that do not otherwise produce immunoglobulin protein, inorder to produce recombinant monoclonal antibodies. Optionally, in oneembodiment, the anti-T. cruzi mouse-human chimeric antibodies of thedisclosure are produced in a Chinese Hamster Ovary (CHO) cell line,which is advantageous in that they can be produced in quantitiessufficient for commercial use. Preferably, the mammalian host cells areCHO cell lines and HEK 293 cell lines. Another preferred host cell isthe B3.2 cell line (e.g., Abbott Laboratories, Abbott BioresearchCenter, Worcester, Mass.), or another dihydrofolate reductase deficient(DHFR-) CHO cell line (e.g., available from Invitrogen Corp., Carlsbad,Calif.).

Alternatively, the DNA encoding the monoclonal antibody or the variableregions thereof can be used to produce chimeric antibodies, humanizedantibodies and antibody fragments by standard methods known in the art.

For example, chimeric monoclonal antibodies can be produced by cloningthe DNA encoding the variable regions of the monoclonal antibody intomammalian expression vector(s) containing antibody heavy and light chainconstant region genes derived from a different host species. Manyeukaryotic antibody expression vectors that are either stably integratedor exist as extrachromosomal elements have been described and are knownto those of ordinary skill in the art. In general, antibody expressionvectors are plasmids comprising the gene encoding the heavy chainconstant region and/or the gene encoding the light chain constantregion, an upstream enhancer element and a suitable promoter.

A wide variety of expression control sequences may be used in thepresent disclosure. Such useful expression control sequences include theexpression control sequences associated with structural genes of theforegoing expression vectors as well as any sequence known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. Examples of suitable controlsequences for directing transcription in mammalian cells include theearly and late promoters of SV40 and adenovirus, for example, theadenovirus major late promoter, the MT-1 (metallothionein gene)promoter, the human cytomegalovirus immediate-early gene promoter (CMV),the human elongation factor 1α (EF-1α) promoter, the Drosophila minimalheat shock protein 70 promoter, the Rous Sarcoma Virus (RSV) promoter,the human ubiquitin C (UbC) promoter, the human growth hormoneterminator, SV40 or adenovirus E1b region polyadenylation signals andthe Kozak consensus sequence (Kozak, J Mol Biol., 196:947-50 (1987)).

For example, for human constant regions, the antibody expression vectorcan comprise the human IgG1 (human Cγ1) and human kappa constant region(human Cκ) genes and the immunoglobulin H chain enhancer element. Thevector can also contain a bacterial origin of replication and selectionmarker. Optional inclusion of a selection marker, as is known in theart, allows for selection and amplification under defined growthconditions, for example the dihydrofolate reductase (DHFR) gene providesfor selection and amplification in mammalian cells with methotrexate.Construction of a vector appropriate for antibody expression startingfrom a commercial mammalian expression vector, can be readily achievedby the skilled technician. As described herein various vectors includingpBV, pJV, and pBOS vectors, as well as variety of intermediary vectorsand plasmids can be employed for antibody production. pBV, pJV, and pBOSvectors were acquired from Abbott Bioresearch Center (Worcester, Mass.).Other similar plasmids and vectors are commercially available and/orreadily constructed.

Introduction of the expression construct(s) into appropriate host cellsresults in production of complete chimeric antibodies of a definedspecificity (see, for example, Morrison, S. L. et al., Proc. Natl. Acad.Sci. USA 81: 6851-6855 (1984)). The heavy and light chain codingsequences can be introduced into the host cell individually on separateplasmids or together on the same vector.

Depending on the vector system used, many different immortalized celllines can serve as suitable hosts, these include, but are not limitedto, myeloma (for example, X63-Ag8.653), hybridoma (for example,Sp2/0-Ag14), lymphoma, insect cells (for example sf9 cells), humanembryonic kidney cells (for example, HEK 293) and Chinese Hamster Ovary(CHO) cells. The expression constructs can be introduced into the hostcells using a variety of techniques known in the art, including but notlimited to, calcium phosphate precipitation, protoplast fusion,lipofection, retrovirus-derived shuttle vectors, and electroporation.

Chimeric antibodies and antibody fragments can also be produced in otherexpression systems including, but not limited to, baculovirus, yeast,bacteria (such as E. coli), and in vitro in cell-free systems, such asrabbit reticulocyte lysate.

The recombinant antibody can be isolated from the host cells by standardimmunoglobulin purification procedures such as, for example, cross-flowfiltration, ammonium sulphate precipitation, protein A chromatography,hydroxylapatite chromatography, gel electrophoresis, dialysis, affinitychromatography, or combinations thereof.

Alternatively, antibody fragments can be generated from a purifiedantibody preparation by conventional enzymatic methods, for example,F(ab′)₂ fragments can be produced by pepsin cleavage of the intactantibody, and Fab fragments can be produced by briefly digesting theintact antibody with papain.

Recombinant bispecific and heteroconjugate antibody fragments havingspecificities for at least two different antigens can be prepared asfull length antibodies or as antibody fragments (such as F(ab′)₂bispecific antibody fragments). Antibody fragments having more than twovalencies (for example, trivalent or higher valency antibody fragments)also are contemplated. Bispecific antibodies, heteroconjugateantibodies, and multi-valent antibodies can be prepared by standardmethods known to those skilled in the art.

2. Monovalent Abs

Monovalent Abs do not cross-link each other. One method involvesrecombinant expression of Ig light chain and modified heavy chain. Heavychain truncations generally at any point in the Fc region prevents heavychain cross-linking. Alternatively, the relevant cysteine residues aresubstituted with another amino acid residue or are deleted, preventingcrosslinking by disulfide binding. In vitro methods are also suitablefor preparing monovalent Abs. Abs can be digested to produce fragments,such as Fab (Harlow and Lane, 1988, supra; Harlow and Lane, 1999,supra).

3. Humanized and Human Abs

Humanized forms of non-human Abs that bind a TCA are chimeric Igs, Igchains or fragments (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of Abs) that contain minimal sequencederived from non-human Ig.

Generally, a humanized antibody has one or more amino acid residuesintroduced from a non-human source. These non-human amino acid residuesare often referred to as “import” residues that are typically taken froman “import” variable domain. Humanization is accomplished bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody (Jones, P. T., et al. 1986. Replacing thecomplementarity-determining regions in a human antibody with those froma mouse. Nature. 321:522-5; Riechmann, L., et al. 1988. Reshaping humanantibodies for therapy. Nature. 332:323-7; Verhoeyen, M., et al. 1988.Reshaping human antibodies: grafting an antilysozyme activity. Science.239:1534-6). Such “humanized” Abs are chimeric Abs (Cabilly et al.,1989), wherein substantially less than an intact human variable domainhas been substituted by the corresponding sequence from a non-humanspecies. In practice, humanized Abs are typically human Abs in whichsome CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent Abs. Humanized Abs include humanIgs (recipient antibody) in which residues from a complementarydetermining region (CDR) of the recipient are replaced by residues froma CDR of a non-human species (donor antibody), such as mouse, rat orrabbit, having the desired specificity, affinity and capacity. In someinstances, corresponding non-human residues replace Fv frameworkresidues of the human Ig. Humanized Abs can include residues that arefound neither in the recipient antibody nor in the imported CDR orframework sequences. In general, the humanized antibody containssubstantially all of at least one, and typically two, variable domains,in which most if not all of the CDR regions correspond to those of anon-human Ig and most if not all of the FR regions are those of a humanIg consensus sequence. The humanized antibody optimally also comprisesat least a portion of an Ig constant region (Fc), typically that of ahuman Ig (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).

Human Abs can also be produced using various techniques, including phagedisplay libraries (Hoogenboom, H. R., et al. 1991. Multi-subunitproteins on the surface of filamentous phage: methodologies fordisplaying antibody (Fab) heavy and light chains. Nucleic Acids Res.19:4133-7; Marks, J. D., et al. 1991. By-passing immunization. Humanantibodies from V-gene libraries displayed on phage. J Mol Biol.222:581-97) and human mAbs (Boerner, P., et al. 1991. Production ofantigen-specific human monoclonal antibodies from in vitro-primed humansplenocytes. J Immunol. 147:86-95; Reisfeld, R. A., and S. Sell. 1985.Monoclonal antibodies and cancer therapy: Proceedings of the Roche-UCLAsymposium held in Park City, Utah, Jan. 26-Feb. 2, 1985. Alan R. Liss,New York. 609 pp). Introducing human Ig genes into transgenic animals inwhich the endogenous Ig genes have been partially or completelyinactivated can be exploited to synthesize human Abs. Upon challenge,human antibody production is observed, which closely resembles that seenin humans in all respects, including gene rearrangement, assembly, andantibody repertoire (Fishwild, D. M., et al. 1996. High-avidity humanIgG kappa monoclonal antibodies from a novel strain of minilocustransgenic mice. Nat Biotechnol. 14:845-51; Lonberg and Huszar, 1995;Lonberg et al., 1994; Marks et al., 1992; Lonberg, N., and R. M. Kay.U.S. Pat. No. 5,569,825. 1996; Lonberg, N., and R. M. Kay. U.S. Pat. No.5,633,425. 1997a; Lonberg, N., and R. M. Kay. U.S. Pat. No. 5,661,016.1997b; Lonberg, N., and R. M. Kay. U.S. Pat. No. 5,625,126. 1997c;Surani, A., et al. U.S. Pat. No. 5,545,807. 1996).

3. Bi-Specific Abs

Bi-specific mAbs bind at least two different antigens. For example, abinding specificity is a TCA; the other is for any antigen of choice.

The recombinant production of bi-specific Abs is often achieved byco-expressing two Ig heavy-chain/light-chain pairs, each havingdifferent specificities. The random assortment of these Ig heavy andlight chains in the resulting hybridomas (quadromas) produce a potentialmixture of ten different antibody molecules, of which only one has thedesired bi-specific structure. The desired antibody can be purifiedusing affinity chromatography or other techniques (Traunecker, A., etal. 1991. Myeloma based expression system for production of largemammalian proteins. Trends Biotechnol. 9:109-13; Wabl, M., J. Berg, andE. Lotscher. WO 93/08829. 1993).

To manufacture a bi-specific antibody, variable domains with the desiredantibody-antigen combining sites are fused to Ig constant domainsequences (Suresh, M. R., A. C. Cuello, and C. Milstein. 1986.Bispecific monoclonal antibodies from hybrid hybridomas. MethodsEnzymol. 121:210-28). The fusion is usually with an Ig heavy-chainconstant domain, comprising at least part of the hinge, CH2, and CH3regions. The first heavy-chain constant region (CH1) containing the sitenecessary for light-chain binding is in at least one of the fusions.DNAs encoding the Ig heavy-chain fusions and, if desired, the Ig lightchain, are inserted into separate expression vectors and areco-transfected into a suitable host organism.

The interface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers that are recovered fromrecombinant cell culture (Carter, P., L. et al. WO 96/27011. 1996). Inthis method, one or more small amino acid side chains from the interfaceof the first antibody molecule are replaced with larger side chains(e.g., tyrosine or tryptophan). Compensatory “cavities” of identical orsimilar size to the large side chain(s) are created on the interface ofthe second antibody molecule by replacing large amino acid side chainswith smaller ones (e.g., alanine or threonine). This mechanism increasesthe yield of the heterodimer over unwanted end products, such ashomodimers.

Bi-specific Abs can be prepared as full length Abs or antibody fragments(e.g., Fab′₂ bi-specific Abs). One technique to generate bi-specific Absexploits chemical linkage. Intact Abs can be proteolytically cleaved togenerate Fab′₂ fragments (Brennan, M., et al. 1985. Preparation ofbispecific antibodies by chemical recombination of monoclonalimmunoglobulin G1 fragments. Science. 229:81-3). Fragments are reducedwith a dithiol complexing agent, such as sodium arsenite, to stabilizevicinal dithiols and prevent intermolecular disulfide formation. Thegenerated Fab′ fragments are then converted to thionitrobenzoate (TNB)derivatives. One of the Fab′-TNB derivatives is then reconverted to theFab′-thiol by reduction with mercaptoethylamine and is mixed with anequimolar amount of the other Fab′-TNB derivative to form thebi-specific antibody.

Fab′ fragments can be directly recovered from E. coli and chemicallycoupled to form bi-specific Abs. For example, fully humanizedbi-specific Fab′₂ Abs can be produced (Shalaby, M. R., et al. 1992.Development of humanized bispecific antibodies reactive with cytotoxiclymphocytes and tumor cells overexpressing the HER2 protooncogene. J ExpMed. 175:217-25). Each Fab′ fragment is separately secreted from E. coliand directly coupled chemically in vitro, forming the bi-specificantibody.

Various techniques for making and isolating bi-specific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, leucine zipper motifs can be exploited(Kostelny, S. A., et al. 1992. Formation of a bispecific antibody by theuse of leucine zippers. J Immunol. 148:1547-53). Peptides from the Fosand Jun polypeptides are linked to the Fab′ portions of two differentAbs by gene fusion. The antibody homodimers are reduced at the hingeregion to form monomers and then re-oxidized to form antibodyheterodimers. This method can also produce antibody homodimers.“Diabody” technology provides an alternative method to generatebi-specific antibody fragments (Holliger et al., 1993). The fragmentsconsist of a heavy-chain V_(H) connected to a light-chain V_(L) by alinker that is too short to allow pairing between the two domains on thesame chain. The V_(H) and V_(L) domains of one fragment are forced topair with the complementary V_(L) and V_(H) domains of another fragment,forming two antigen-binding sites. Another strategy for makingbi-specific antibody fragments is the use of single-chain Fv (sFv)dimers (Gruber, M., et al. 1994. Efficient tumor cell lysis mediated bya bispecific single chain antibody expressed in Escherichia coli. JImmunol. 152:5368-74). Abs with more than two valencies can also bemade, such as tri-specific Abs (Tutt, A., et al. 1991. TrispecificF(ab′)3 derivatives that use cooperative signaling via the TCR/CD3complex and CD2 to activate and redirect resting cytotoxic T cells. JImmunol. 147:60-9). Exemplary bi-specific Abs can bind to two differentepitopes on a given TCA.

H. Testing of Recombinant Antibodies

The ability of the recombinant antibody to specifically bind to thetarget T. cruzi antigen can be assessed by standard immunologicaltechniques (see, for example, Current Protocols in Immunology, Coligan,J. E., et al. (ed.), J. Wiley & Sons, New York, N.Y.). For example, byradioimmunoassay (RIA) or enzyme immunoassay (EIA or ELISA). In oneembodiment of the present disclosure, the recombinant antibodydemonstrates substantially the same specificity as the monoclonalantibody from which the antigen-binding domains are derived.

The recombinant antibodies optionally can also be tested for theirbinding affinity to the target T. cruzi antigen by measuring theequilibrium dissociation constant (K_(D)) by standard techniques. In oneembodiment of the present disclosure, the recombinant antibodies (e.g.,chimeric antibodies) have a K_(D) less than about 1 μM. In anotherembodiment, the recombinant antibodies (e.g., chimeric antibodies) havea K_(D) less than about 100 nM.

Other standard tests also can be done on the antibodies, for example,the pI value of the antibodies can be obtained.

Optionally, the recombinant antibodies (e.g., chimeric antibodies) aresubjected to epitope mapping procedures to identify the region of thetarget antigen to which they bind. A variety of methods of epitopemapping are known in the art (see, for example, Current Protocols inImmunology, Coligan, J. E., et al. (ed.), J. Wiley & Sons, New York,N.Y.) and include, for example, phage and yeast display methods. Phageand yeast display methods can also be combined with random mutagenesistechniques in order to more precisely map the residues of the targetantigen involved in antibody binding (see, for example, Chao, G., etal., J. Mol. Biol., 10:539-50 (2004)).

In one embodiment of the present disclosure, the residues of the targetantigen to which the recombinant antibodies bind are identified by atechnique that combines scanning alanine mutagenesis with yeast display.The technique generally involves the preparation of a series ofoligonucleotides encoding peptides each representing the target regionof the antigen and in which each individual amino acid in this regionwas sequentially substituted by alanine. The target region of theantigen is determined either from the antigen used in the initialimmunization to prepare the parent monoclonal antibody, or from apreliminary “low-resolution” screening using yeast or phage display. Awildtype version of the antigen is used as a control. Eacholigonucleotide is cloned into an appropriate yeast display vector andeach alanine mutant transformed into a suitable host, such as E. coli.Plasmid DNA is extracted and sequenced and clones are selected based onsequencing. Yeast display vectors are known in the art and arecommercially available (for example, pYD1 available from InvitrogenCorp., Carlsbad, Calif., USA).

The selected clones are then transformed into Saccharomyces cerevesiaecells, for example, EBY100 cells (Invitrogen Corp.), and individualyeast clones cultured and induced for peptide expression. The inducedyeast cells expressing the alanine mutants on the cell surface areincubated with the recombinant antibody and bound antibody is detectedby conventional methods, for example using a labeled secondary antibody.Key residues in the target antigenic region can then be determined basedon the identification of alanine mutants unable to bind to therecombinant antibody. A loss of antibody binding activity indicates thatthe mutant includes an alanine residue at a position that forms part ofthe epitope for the recombinant antibody.

I. Uses of Recombinant Antibodies

The recombinant antibodies of the present disclosure are suitable foruse, for example, as diagnostic reagents for the detection of T. cruzi,and/or as standardization reagents, positive control reagents orcalibrator reagents in assays or kits for the detection of T. cruziantibodies in place of traditional plasma or serum. Standardizationreagents can be used, for example, to establish standard curves forinterpolation of antibody concentration. Positive controls can be usedto establish assay performance characteristics and/or quantitate andmonitor the integrity of the antigen(s) used in the assay. The presentdisclosure also provides for the use of a plurality of the recombinantantibodies, each recombinant antibody capable of specifically binding toa different T. cruzi antigen, as standardized antibody sensitivitypanels. Such sensitivity panels can be used, for example, in place oftraditional plasma or serum for quality control of T. cruzi antibodydetection kits, to establish assay performance characteristics and/orquantitate and monitor the integrity of the antigen(s) used in theassay. The present disclosure also contemplates the use of therecombinant antibodies in the treatment or prevention of a T. cruziinfection.

One embodiment of the present disclosure thus provides for animmunodiagnostic reagent comprising one or more recombinant antibodies,each capable of specifically binding a diagnostically relevant region ofa T. cruzi protein.

In one embodiment of the present disclosure, the immunodiagnosticreagent comprises a plurality of (for example, two or more) recombinantantibodies each capable of detecting a different T. cruzi antigen.

The immunodiagnostic reagent can be tailored for a specific end use byappropriate selection of the recombinant antibodies it comprises, thusmaking the immunodiagnostic reagent compatible with a number of existingT. cruzi detection assay formats and kits. Tailoring theimmunodiagnostic reagent in this manner also allows the reagent to beoptimized for detection of certain stages of T. cruzi infection.

The present disclosure further provides for a method of standardizing T.cruzi antibody detection assays using an immunodiagnostic reagentcomprising a plurality of recombinant antibodies, each capable ofspecifically binding to a different TCA, as a sensitivity panel.

The present disclosure additionally provides for a method for detectingthe presence of TCAs which comprises contacting a test sample suspectedof containing TCAs with an immunodiagnostic reagent comprising one ormore recombinant antibodies, each capable of specifically binding a TCA,under conditions that allow formation of recombinant antibody:antigencomplexes and detecting any recombinant antibody:antigen complexesformed.

The present disclosure also encompasses a method for detecting thepresence of T. cruzi antibodies which comprises contacting a test samplesuspected of containing T. cruzi antibodies with one or more antigensspecific for the T. cruzi antibodies, under conditions that allowformation of antigen/antibody complexes, detecting the antigen:antibodycomplexes, and using an immunodiagnostic reagent comprising one or morerecombinant antibodies, each capable of specifically binding one of theantigens used in the method, as a positive control or standardizationreagent.

The immunodiagnostic reagents of the present disclosure are suitable foruse with assays and kits monitoring T. cruzi responses in man as well asother vertebrate species susceptible to T. cruzi infection and capableof generating an antibody response thereto. The immunodiagnosticreagents thus have human medical as well as veterinary applications.

The present disclosure also encompasses the use of the recombinantantibodies and variable regions described herein in directed molecularevolution technologies such as phage display technologies, and bacterialand yeast cell surface display technologies, in order to produce novelrecombinant antibodies in vitro (See, for example, Johnson et al.,Current Opinion in Structural Biology 3:564 (1993) and Clackson et al.,Nature 352:624 (1991)).

Optionally the immunodiagnostic reagent of the disclosure, e.g., thechimeric antibodies, can be used in commercial platform immunoassays.

J. Kits Comprising Recombinant Antibodies

The present disclosure further provides for therapeutic, diagnostic andquality control kits comprising one or more recombinant antibodies ofthe disclosure.

One aspect of the present disclosure provides diagnostic kits for thedetection of T. cruzi. The kits comprise one or more recombinantantibodies of the present disclosure. The recombinant antibodies can beprovided in the kit as detection reagents, either for use to captureand/or detect T. cruzi antigens or for use as secondary antibodies forthe detection of antigen:antibody complexes. Alternatively, therecombinant antibodies can be provided in the kit as a positive controlreagent, a standardization reagent, calibration reagent or a sensitivitypanel. In various embodiments, the diagnostic kit can further comprisereagents for detection of T. cruzi antigens or reagents for thedetection of T. cruzi antibodies. In one embodiment, the presentdisclosure provides a diagnostic kit comprising reagents for detectionof T. cruzi antibodies, including one or more antigens specific for theT. cruzi antibodies, and a positive control or standardization reagentcomprising one or more recombinant antibodies of the disclosure, eachcapable of specifically binding one of the one or more antigens includedin the kit.

Thus, the present disclosure further provides for diagnostic and qualitycontrol kits comprising one or more antibodies of the disclosure.Optionally the assays, kits and kit components of the disclosure areoptimized for use on commercial platforms (e g, immunoassays on thePrism®, AxSYM®, ARCHITECT® and EIA (Bead) platforms of AbbottLaboratories, Abbott Park, Ill., as well as other commercial and/or invitro diagnostic assays). Additionally, the assays, kits and kitcomponents can be employed in other formats, for example, onelectrochemical or other hand-held or point-of-care assay systems. Thepresent disclosure is, for example, applicable to the commercial AbbottPoint of Care (i-STAT®, Abbott Laboratories, Abbott Park, Ill.)electrochemical immunoassay system that performs sandwich immunoassaysfor several cardiac markers, including TnI, CKMB and BNP Immunosensorsand methods of operating them in single-use test devices are described,for example, in US Patent Applications 20030170881, 20040018577,20050054078 and 20060160164 which are incorporated herein by reference.Additional background on the manufacture of electrochemical and othertypes of immunosensors is found in U.S. Pat. No. 5,063,081 which is alsoincorporated by reference for its teachings regarding same.

Optionally the kits include quality control reagents (e.g., sensitivitypanels, calibrators, and positive controls). Preparation of qualitycontrol reagents is well known in the art, and is described, e.g., on avariety of immunodiagnostic product insert sheets. Sensitivity panelmembers optionally can be prepared in varying amounts containing, e.g.,known quantities of antibody ranging from “low” to “high”, e.g., byspiking known quantities of the antibodies according to the disclosureinto an appropriate assay buffer (e.g., a phosphate buffer). Thesesensitivity panel members optionally are used to establish assayperformance characteristics, and further optionally are usefulindicators of the integrity of the immunoassay kit reagents, and thestandardization of assays.

The antibodies provided in the kit can incorporate a detectable label,such as a fluorophore, radioactive moiety, enzyme, biotin/avidin label,chromophore, chemiluminescent label, or the like, or the kit may includereagents for labeling the antibodies or reagents for detecting theantibodies (e.g., detection antibodies) and/or for labeling the antigensor reagents for detecting the antigen. The antibodies, calibratorsand/or controls can be provided in separate containers or pre-dispensedinto an appropriate assay format, for example, into microtiter plates.

The kits can optionally include other reagents required to conduct adiagnostic assay or facilitate quality control evaluations, such asbuffers, salts, enzymes, enzyme co-factors, substrates, detectionreagents, and the like. Other components, such as buffers and solutionsfor the isolation and/or treatment of a test sample (e.g., pretreatmentreagents), may also be included in the kit. The kit may additionallyinclude one or more other controls. One or more of the components of thekit may be lyophilized and the kit may further comprise reagentssuitable for the reconstitution of the lyophilized components.

The various components of the kit optionally are provided in suitablecontainers. As indicated above, one or more of the containers may be amicrotiter plate. The kit further can include containers for holding orstoring a sample (e.g., a container or cartridge for a blood or urinesample). Where appropriate, the kit may also optionally contain reactionvessels, mixing vessels and other components that facilitate thepreparation of reagents or the test sample. The kit may also include oneor more instruments for assisting with obtaining a test sample, such asa syringe, pipette, forceps, measured spoon, or the like.

The kit further can optionally include instructions for use, which maybe provided in paper form or in computer-readable form, such as a disc,CD, DVD or the like.

K. Adaptation of Kits

The kit (or components thereof), as well as the method of determiningthe detecting the presence or concentration of T. cruzi antigens in atest sample by an assay using the components and methods describedherein, can be adapted for use in a variety of automated andsemi-automated systems (including those wherein the solid phasecomprises a microparticle), as described, e.g., in U.S. Pat. Nos.5,089,424 and 5,006,309, and as commercially marketed, e.g., by AbbottLaboratories (Abbott Park, Ill.) as ARCHITECT®.

Some of the differences between an automated or semi-automated system ascompared to a non-automated system (e.g., ELISA) include the substrateto which the first specific binding partner (e.g., T. cruzi captureantibody) is attached (which can impact sandwich formation and analytereactivity), and the length and timing of the capture, detection and/orany optional wash steps. Whereas a non-automated format such as an ELISAmay require a relatively longer incubation time with sample and capturereagent (e.g., about 2 hours) an automated or semi-automated format(e.g., ARCHITECT®, Abbott Laboratories) may have a relatively shorterincubation time (e.g., approximately 18 minutes for ARCHITECT®).Similarly, whereas a non-automated format such as an ELISA may incubatea detection antibody such as the conjugate reagent for a relativelylonger incubation time (e.g., about 2 hours), an automated orsemi-automated format (e.g., ARCHITECT®) may have a relatively shorterincubation time (e.g., approximately 4 minutes for the ARCHITECT®).

Other platforms available from Abbott Laboratories include, but are notlimited to, AxSYM®, IMx® (see, e.g., U.S. Pat. No. 5,294,404, which ishereby incorporated by reference in its entirety), PRISM®, EIA (bead),and Quantum™ II, as well as other platforms. Additionally, the assays,kits and kit components can be employed in other formats, for example,on electrochemical or other hand-held or point-of-care assay systems.The present disclosure is, for example, applicable to the commercialAbbott Point of Care (i-STAT®, Abbott Laboratories) electrochemicalimmunoassay system that performs sandwich immunoassays Immunosensors andtheir methods of manufacture and operation in single-use test devicesare described, for example in, U.S. Pat. No. 5,063,081, U.S. Pat. App.Pub. No. 2003/0170881, U.S. Pat. App. Pub. No. 2004/0018577, U.S. Pat.App. Pub. No. 2005/0054078, and U.S. Pat. App. Pub. No. 2006/0160164,which are incorporated in their entireties by reference for theirteachings regarding same.

In particular, with regard to the adaptation of a T. cruzi antigen assayto the I-STAT® system, the following configuration is preferred. Amicrofabricated silicon chip is manufactured with a pair of goldamperometric working electrodes and a silver-silver chloride referenceelectrode. On one of the working electrodes, polystyrene beads (0.2 mmdiameter) with immobilized capture antibody are adhered to a polymercoating of patterned polyvinyl alcohol over the electrode. This chip isassembled into an I-STAT® cartridge with a fluidics format suitable forimmunoassay. On a portion of the wall of the sample-holding chamber ofthe cartridge there is a layer comprising the second detection antibodylabeled with alkaline phosphatase (or other label). Within the fluidpouch of the cartridge is an aqueous reagent that includes p-aminophenolphosphate.

In operation, a sample suspected of containing a T. cruzi antigen isadded to the holding chamber of the test cartridge and the cartridge isinserted into the I-STAT® reader. After the second antibody (detectionantibody) has dissolved into the sample, a pump element within thecartridge forces the sample into a conduit containing the chip. Here itis oscillated to promote formation of the sandwich between the T. cruziantigen, T. cruzi capture antibody, and the labeled detection antibody.In the penultimate step of the assay, fluid is forced out of the pouchand into the conduit to wash the sample off the chip and into a wastechamber. In the final step of the assay, the alkaline phosphatase labelreacts with p-aminophenol phosphate to cleave the phosphate group andpermit the liberated p-aminophenol to be electrochemically oxidized atthe working electrode. Based on the measured current, the reader is ableto calculate the amount of T. cruzi antigen in the sample by means of anembedded algorithm and factory-determined calibration curve.

It further goes without saying that the methods and kits as describedherein necessarily encompass other reagents and methods for carrying outthe immunoassay. For instance, encompassed are various buffers such asare known in the art and/or which can be readily prepared or optimizedto be employed, e.g., for washing, as a conjugate diluent, and/or as acalibrator diluent. An exemplary conjugate diluent is ARCHITECT®conjugate diluent employed in certain kits (Abbott Laboratories, AbbottPark, Ill.) and containing 2-(N-morpholino)ethanesulfonic acid (MES), asalt, a protein blocker, an antimicrobial agent, and a detergent. Anexemplary calibrator diluent is ARCHITECT® human calibrator diluentemployed in certain kits (Abbott Laboratories, Abbott Park, Ill.), whichcomprises a buffer containing MES, other salt, a protein blocker, and anantimicrobial agent.

To gain a better understanding of the disclosure described herein, thefollowing examples are set forth. It will be understood that theseexamples are intended to describe illustrative embodiments of thedisclosure and are not intended to limit the scope of the disclosure inany way.

EXAMPLES Example 1 Cell Lines Producing Antibodies Against T. cruziAntigen FP3 (Chagas FP3 12-392-150-110)

In this example, a hybridoma cell line that produces mAbs that recognizeand bind Chagas FP3 recombinant antigen was produced. Mice wereimmunized with the FP3 recombinant antigen (SEQ ID NO.:2), the anti-FP3antibody-producing mice euthanized, spleen cells harvested and fusedwith myeloma cells, and mAb anti-FP3 hybridoma cell lines were isolated.The resulting cell line HBFP3 was produced.

Immunogen Preparation

The Chagas FP3 antigen cell line was provided by Dr. Louis Kirchoff slaboratory of the University of Iowa, for a seed bank in Lake County,Illinois. The cDNA sequence encoding this antigen (SEQ ID NO.:1) wascloned into the pET expression vectors under the control of T7 promoterand expressed in suitable E. coli host cells [BLR(DE3)pLysS orBL21(DE3)]. The T7 RNA polymerase was encoded by the lambda DE3 lysogeninserted into the host bacterial genome and under the control of thelacUV5 promoter. Isopropyl β-D-thiogalactopyranoside (IPTG) was added tothe cells to induce T7 RNA polymerase expression, which in turn bound tothe T7 promoter and resulted in the expression of the cloned gene.Plates of the transformed cells were streaked to isolate a single colonyand cell banks were prepared. Subsequently, the E. coli was grown, and acell paste was prepared for purification.

First, the recombinant FP3 antigen was purified from clarifiedsupernatant by recirculating the clarified supernatant during loading.Second, spuriously bound contaminants where removed from the ImmobilizedMetal Affinity Chromatography (IMAC) column by washing the affinitycolumn with a high salt buffer. Third, the His-tagged (amino end)recombinant FP3 polypeptide was eluted from the column by competitivelyremoving His-tagged antigen with imidazole. Subsequently, the elutedproteins were fractioned and analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Fractions thatcontained the recombinant FP3 antigen without significant contaminationwere pooled and concentrated. After concentration, the FP3 antigen wasfurther purified by size exclusion chromatography using a 2000 mlSephacryl S-300 sizing column, analyzed by SDS-PAGE and concentrated.

Animal Immunization

RBf/dnJ female mice (all mice were obtained from Jackson Laboratories;Bar Harbor, Me.) were immunized twice with purified Chagas FP4recombinant antigen (containing the target FP3 sequence) and once withpurified Chagas FP3 recombinant antigen, using the Freunds AdjuvantSystem, prior to checking the antisera for sufficient titer. Theinoculum was prepared by diluting the antigen in 0.9% sodium chlorideand emulsifying with an equal volume of adjuvant. At weeks 0 and 3, a 20μg boost of FP4 (containing the target FP3 sequence) was administered tothe mice. At week 6, a 10 μg boost of FP3 was administered to the mice.Freunds Complete Adjuvant was used for the primary boost deliveredsubcutaneously, and Freunds Incomplete Adjuvant was used for the next 2intradermal boosts. Two weeks after the third boost, a sera sample wastaken for a specific anti-T. cruzi titer test, which resulted in theselection of mouse #241 for the fusion experiment. Three days prior tothe fusion, mouse #241 was administered a pre-fusion boost of 5 μg ofthe FP3 recombinant antigen.

Hybridoma Creation (Cell Fusion Experiment)

Hybridomas were developed using the polyethylene glycol (PEG)-mediatedfusion technique described in Galfre et al. (Galfre, G., et al. 1977.Antibodies to major histocompatibility antigens produced by hybrid celllines. Nature. 266:550-2). The RBUdnJ mouse #241 was euthanized threedays after the pre-fusion boost, and the spleen was harvested. TheB-cells were perfused from the spleen, washed and re-suspended in anequal number of SP2/0 myeloma cells (ATTC deposit CRL-1581). The totalcells were pelleted, and the fusion was performed with 1 ml ofpolyethylene glycol (PEG) and cultured at 37° C. in HAT-supplementedGIBCO® Hybridoma Serum Free Medium (H-SFM; Invitrogen Corp., Carlsbad,Calif.) with 10% fetal bovine serum (FBS; Hyclone; Logan, Utah). Cellswere plated into 96-well tissue culture plates and incubated in ahumidified 37° C. incubator. The hybrids were tested 10-14 days laterfor anti-T. cruzi FP3 reactivity in a microtiter enzyme immunoassay(EIA). The results indicated hybrid 12-392 secreted anti-FP3 specificantibody.

Hybridoma Cloning and Subcloning

Hybridoma 12-392 was selected for limiting dilution cloning. The cellswere suspended and then serially diluted 10⁴, 10⁵ and 10⁶ into 20 ml ofH-SFM with 10% FBS. Each dilution was plated into a 96-well tissueculture plate with 0.2 ml cell suspension per well. The plates wereincubated for 10-14 days at 37° C. in a humidified incubator. As growthbecame apparent, the supernates were tested in an anti-FP3 microtiterEIA that resulted in the selection of clone 12-392-150.

Clone 12-392-150 was selected for subcloning using fluorescenceactivated cell sorting (FACS). A cell suspension was stained with goatanti-mouse-Alexa Fluor 488 (Invitrogen Corp., Carlsbad, Calif.). Singlecell isolates from the top 5-8% of this stained cell population weredeposited in a 96-well tissue culture plate with 0.2 ml of H-SFM with10% FBS. The plates were incubated for 10-14 days at 37° C. in ahumidified incubator. As growth became apparent, the supernates weretested in an anti-FP3 microtiter EIA that resulted in the selection ofclone 12-392-150-110 (HBFP3).

HBFP3 was expanded in tissue culture to a 850 cm² roller bottle, cellpassage 5, in H-SFM with 10% FBS. The pass 5 cell suspension waspelleted, re-suspended in freeze medium and dispensed into cryovials.The vials were stored in liquid nitrogen storage tanks.

Example 2 Cell Lines Producing Antibodies Against Chagas TcF RecombinantAntigen (Chagas 9-638-132-115)

Immunogen Source

The purified TcF recombinant antigen (containing the PEP2 sequence) usedfor animal immunizations was obtained from Corixa Corporation (Seattle,Wash.).

Animal Immunization

RBUdnJ female mice were immunized three times with purified Chagas TcFrecombinant antigen, using the Freunds Adjuvant System, prior tochecking the antisera for sufficient titer. The inoculum was prepared bydiluting the antigen in 0.9% sodium chloride and emulsifying with anequal volume of adjuvant. At weeks 0, 6, and 12, a 20 μg boost of TcFwas administered to the mice. Freunds Complete Adjuvant was used for theprimary boost delivered subcutaneously and Freunds Incomplete Adjuvantwas used for the next 2 intradermal boosts. Two weeks after the 3rdboost, a sera sample was taken for a specific anti-T. cruzi titer test,which resulted in the selection of mouse #115 for the fusion experiment.Three days prior to the fusion, mouse #115 was administered a pre-fusionboost of 10 μg of the TcF recombinant antigen and 10 μg of the TcF Pep2peptide.

Hybridoma Creation

Hybridomas were developed using PEG-mediated fusion technique describedin Galfre et al. (Galfre et al., 1977). The RBUdnJ mouse #115 waseuthanized three days after the pre-fusion boost, and the spleen washarvested. The B-cells were perfused from the spleen, washed andre-suspended in an equal number of SP2/0 myeloma cells (ATTC depositCRL-1581). The total cells were pelleted, and the fusion was performedwith 1 ml of PEG and cultured at 37° C. in HAT-supplemented GIBCO® H-SFM(Invitrogen Corp., Carlsbad, Calif.) with 10% FBS (Hyclone, Logan,Utah). Cells were plated into 96-well tissue culture plates andincubated in a humidified 37° C. incubator. The hybrids were tested10-14 days later for anti-T. cruzi Pep2 reactivity in a microtiter EIA.The results indicated hybrid 9-638 secreted anti-Pep2 specific antibody.

Hybridoma Cloning and Subcloning

Hybridoma 9-638 was selected for limiting dilution cloning. The cellswere suspended and then serially diluted 10⁴, 10⁵ and 10⁶ into 20 ml ofH-SFM with 10% FBS. Each dilution was plated into a 96-well tissueculture plate with 0.2 ml cell suspension per well. The plates wereincubated for 10-14 days at 37° C. in a humidified incubator. As growthbecame apparent, the supernates were tested in an anti-Pep2 microtiterEIA, and clone 9-638-132 was selected.

Clone 9-638-132 was selected for subcloning using FACS. A cellsuspension was stained with goat anti-mouse-Alexa Fluor 488. Single cellisolates from the top 1% of this stained cell population were depositedin a 96-well tissue culture plate with 0.2 ml of H-SFM with 10% FBS. Theplates were incubated for 10-14 days at 37° C. in a humidifiedincubator. As growth became apparent, the supernates were tested in ananti-Pep2 microtiter EIA, and clone 9-638-132-115 was selected.

Clone 9-638-132-115 was expanded in tissue culture to a 850 cm² rollerbottle, cell passage 6, in H-SFM with 10% FBS. The pass 5 cellsuspension was pelleted. The pellet was then re-suspended in freezemedium and dispensed into cryovials. The vials were stored in liquidnitrogen storage

Example 3 Cell Lines Producing Antibodies Against Chagas FP10Recombinant Antigen (Chagas 10-745-140)

Immunogen Source

The Chagas FP10 antigen (SEQ ID NO.:6) cell line was obtained from thelaboratory of Dr. Louis Kirchoff, University of Iowa, for a seed bank inLake County.

The cDNA sequence (SEQ ID NO.: 5) encoding this antigen was cloned intothe pET expression vectors, and the cells were processed and recombinantantigen purified as outlined in Example 1.

Animal Immunization

RBf/dnJ female mice were immunized three times with purified Chagas FP10recombinant antigen using the Freunds Adjuvant System prior to checkingthe antisera for sufficient titer. The inoculum was prepared by dilutingthe antigen in 0.9% sodium chloride and emulsifying with an equal volumeof adjuvant. At weeks 0, 3, and 6, a 20 μg boost was administered to themice. Freunds Complete Adjuvant was used for the primary boost deliveredsubcutaneously, and Freunds Incomplete Adjuvant was used for the next 2intradermal boosts. Two weeks after the 3rd boost, a sera sample wastaken for a specific anti-T. cruzi titer test, and mouse #230 wasselected for the fusion experiment. Three days prior to the fusion,mouse #230 was administered a pre-fusion boost consisting of 25 μg ofthe FP10 recombinant antigen and 25 μg of a 14 amino acid syntheticpeptide representing the L-domain of the FP10 recombinant antigen.

Hybridoma Creation

Hybridomas were developed using PEG-mediated fusion technique describedin Galfre et al. (Galfre et al., 1977). The RBUdnJ mouse #230 waseuthanized three days after the pre-fusion boost, and the spleen washarvested. The B-cells were perfused from the spleen, washed andre-suspended in an equal number of SP2/0 myeloma cells (ATTC depositCRL-1581). The total cells were pelleted, and the fusion was performedwith 1 ml of PEG and cultured at 37° C. in HAT-supplemented GIBCO® H-SFM(Invitrogen Corp., Carlsbad, Calif.) with 10% FBS (Hyclone, Logan,Utah). Cells were plated into 96-well tissue culture plates andincubated in a humidified 37° C. incubator. The resulting hybridomaswere tested 10-14 days later for anti-T. cruzi FP10 reactivity in anEIA. A hybridoma secreting anti-T. cruzi FP10 mAb known as 10-745 wasselected.

Hybridoma Cloning

Hybrid 10-745 was selected for a limiting dilution cloning. The cellswere suspended and then serially diluted 10⁴, 10⁵ and 10⁶ into 20 ml ofH-SFM with 10% FBS. Each dilution was plated into a 96-well tissueculture plate with 0.2 ml cell suspension per well. The plates wereincubated for 10-14 days at 37° C. in a humidified incubator. As growthbecame apparent, the supernates were tested in an anti-FP10 microtiterEIA that resulted in the selection of clone 10-745-140.

Clone 10-745-140 was expanded in tissue culture to a T75-flask, cellpassage 2, in IMDM with 10% FBS. The pass 2 cell suspension was pelletedby centrifugation. The pellet was then resuspended in freeze medium anddispensed into appropriately labeled cryovials. The vials were stored inliquid nitrogen storage tanks.

Example 4 Cell Lines Producing Antibodies Against Chagas FRA RecombinantAntigen (Chagas FRA 8-367-171)

Immunogen Source

The T. cruzi antigen cell line containing the FRA region (SEQ ID NO.:8)comprised in the FP6 polypeptide, was obtained from the laboratory ofDr. Louis Kirchoff, University of Iowa, for a seed bank in Lake County.The cDNA sequence encoding this antigen (SEQ ID NO.:7) was cloned intothe pET expression vectors, and the cells were processed and recombinantantigen purified as outlined in Example 1.

Animal Immunizations

BALB/c female mice were immunized three times with purified Chagasrecombinant antigen FP6 using the Freunds Adjuvant System prior tochecking the antisera for sufficient titer. The inoculum was prepared bydiluting the antigen in 0.9% sodium chloride and emulsifying with anequal volume of adjuvant. At weeks 0, 4, and 10, a 10 μg boost wasadministered to the mice. Freunds Complete Adjuvant was used for theprimary boost delivered subcutaneously and Freunds Incomplete Adjuvantwas used for the next 2 intradermal boosts. Two weeks after the 3rdboost, a sera sample was taken for a specific anti-T. cruzi titer test,and mouse #1907 was selected for the fusion experiment. Three days priorto fusion, mouse #1907 was administered a pre-fusion boost consisting of25 μg of the recombinant antigen and 25 μg of a synthetic peptiderepresenting the FRA-domain of the recombinant antigen.

Hybridoma Creation

Hybridomas were developed using PEG-mediated fusion technique describedin Galfre et al. (Galfre et al., 1977). The BALB/c mouse #1907 waseuthanized three days after the pre-fusion boost, and the spleen washarvested. The B-cells were perfused from the spleen, washed andre-suspended in an equal number of SP2/0 myeloma cells (ATTC depositCRL-1581). The total cells were pelleted, and the fusion was performedwith 1 ml of PEG and cultured at 37° C. in HAT-supplemented GIBCO® H-SFM(Invitrogen Corp., Carlsbad, Calif.) with 10% FBS (Hyclone, Logan,Utah). Cells were plated into 96-well tissue culture plates andincubated in a humidified 37° C. incubator. The resulting hybridomaswere tested 10-14 days later for anti-T. cruzi FRA-domain reactivity inan EIA. A hybridoma secreting anti-T. cruzi FRA-domain mAb known as8-367 was selected.

Hybridoma Cloning

Hybrid 8-367 was selected for a limiting dilution cloning. The cellswere suspended and then serially diluted 10⁴, 10⁵ and 10⁶ dilutions into20 ml of H-SFM with 10% FBS. Each dilution was plated into a 96-welltissue culture plate with 0.2 ml cell suspension per well. The plateswere incubated for 10-14 days at 37° C. in a humidified incubator. Asgrowth became apparent, the supernates were tested in an anti-FRAmicrotiter EIA, and clone 8-367-171 was selected.

Clone 8-367-171 was expanded in tissue culture to a T75-flask, cellpassage 3, in IMDM with 10% FBS. The pass 2 cell suspension waspelleted, re-suspended in freeze medium and dispensed into cryovials.The vials were stored in liquid nitrogen storage tanks.

Example 5 Cell Lines Producing Chimeric Anti-T. cruzi FP3 mAbs (ChagasFP3 12-392-150CH02580-104)

In this and the subsequent examples directed towards the creation ofmammalian cell lines that express mouse-human chimeric monoclonalantibodies, the following overall approach was taken. After identifyinghybridoma cells lines that secreted the desired mAbs (such as thehybridomas of Examples 1-4), mRNA was isolated from these cells and theantibody gene sequences identified. The V_(L) and V_(H) sequences werethen cloned into pBOS vectors, which supplied the human antibodyconstant sequences (Mizushima S, Nagata S., “pEF-BOS, a powerfulmammalian expression vector.” Nucleic Acids Res. 1990 Sep. 11;18(17):5322 and US 2005/0227289 (incorporated by reference for itsteachings regarding the use of these vectors and the vectorsthemselves)), which were then co-transfected in a transient expressionsystem to confirm that the resulting chimeric antibodies werefunctional. Upon confirmation, the V_(L) sequences were sub-cloned intopJV, and the V_(H) sequences into pBV; these vectors where then used toconstruct a stable pBJ expression vector. The pJV plasmid was obtainedfrom Abbott Laboratories (Abbott Bioresearch Center, Worcester, Mass.)and comprises a SV40 promoter, a murine DHFR gene, an enhancer, apromoter, and a lambda stuffer. The pBV plasmid (also obtained fromAbbott Laboratories, Abbott Bioresearch Center, Worcester, Mass.)comprises an enhancer, a promoter, and a lambda stuffer. Chinese HamsterOvary (CHO) cells were then transfected with pBJ, stable transfectantsselected, and the secreted antibodies tested again. FIG. 1 shows aschematic summary of the chimeric antibodies, where the murine variableregion genes (antigen binding portion) are transferred into vectorswhere the human constant region genes are appended.

Identification of Mouse V_(H) and V_(L) Sequences

Hybridoma cell line HBFP3 (Example 1) was cultured in H-SFM to obtain˜5×10⁶ cells for mRNA purification according to standard mRNA extractionprotocols. The purified mRNA was used as a template with a mouse Igprimer set (Novagen (EMD Biosciences, Inc.); Madison, Wis.) in an RT-PCRreaction. Positive PCR products were observed from the heavy chain (H)primers B and C (HB and HC clones) and from the light chain (L) primersA, B, C, and G (LA, LB, LC, and LG clones). All positive PCR productswere gel-purified and cloned into pCR TOPO 2.1 TA vector (InvitrogenCorp., Carlsbad, Calif.). The plasmid DNA was purified from transformedbacterial cells and the V_(H) or V_(L) inserts were confirmed by EcoRIdigestion for each RT-PCR reaction that generated appropriately sizedproducts. The correct V_(H) or V_(L) gene sequence was selected aftersequence alignments confirmed a consensus sequence among the clones.Chagas TOPO-TA clone HB1 contained the correct V_(H) gene sequence, andChagas TOPO-TA clone LG3 contained the correct V_(L) gene sequence.

Cloning Murine V_(H) and V_(L) Genes into pBOS Vectors

A pair of PCR primers containing a partial Kappa signal sequence with anNru I site on the 5′-primer, and a BsiW I site on the 3′-primer was usedto amplify the mouse V_(L) gene from TOPO clone LG3. Additionally, apair of primers containing a partial heavy chain signal sequence and anNru I site on the 5′-primer, and Sal I site on 3′-primer was used toamplify the mouse V_(H) gene from TOPO clone HB1. The V_(L) PCR productwas digested with Nru I and BsiW I restriction enzymes and ligated intopBOS-hCk vector digested with the same enzymes. The V_(H) PCR productwas digested with Nru I and Sal I restriction enzymes and ligated intopBOS-hCgl vector digested with the same enzymes. Plasmids from a numberof transformed bacterial colonies were sequenced to confirm the presenceof either the Chagas V_(H) or V_(L) gene in their respective vectors.Chagas 12-392-150 V_(H) _(_)pBOS-H clone 4 and Chagas 12-392-150 V_(L)_(_)pBOS-L clone 5 were deemed correct.

Chimeric mAb Production and Functional Confirmation

Endotoxin-free plasmid preparations of Chagas 12-392-150 V_(H)_(_)pBOS-H clone 1 and Chagas 12-392-150 V_(L) _(_)pBOS-L clone 4 wereused for transient transfection into COS 7L cells by electroporation(GENE PULSER®, Bio-Rad; Hercules, Calif.). The transfected cells wereincubated at 37° C. in a 5% CO₂ incubator for three days. The chimericantibody produced by the COS 7L cells were harvested by centrifugationat 4000 rpm for 20 minutes and then purified using a protein A affinitycolumn (POROS A; Applied Biosystems; Foster City, Calif.). To confirmactivity, the harvested antibody was assayed using surface plasmonresonance on a BIACORE® instrument (Biacore (GE Healthcare); Piscataway,N.J.).

CHO Cell Line Stable Expression Vector Cloning

Chagas 12-392-150 V_(H) _(_)pBOS-H clone 1 and Chagas 12-392-150 V_(L)_(_)pBOS-L clone 4 were used to construct a plasmid to generate astable, transfected CHO cell line. First, Srf I and Not I were used toisolate the V_(H)-CH and V_(L)-CL genes from the pBOS vectors; thesefragments were then cloned into pBV or pJV vectors, respectively. Bothvectors were acquired from Abbott Bioresearch Center (Worcester, Mass.)and contained regulatory sequences needed for the expression of theantibody genes. The resulting pBV and pJV clones were analyzed by SrfI/Not I restriction enzyme digestion and sequenced to determine Chagas10-745 V_(H) _(_)pBV clone 4 and Chagas 12-392-150_pJV clone 1 werecorrect. Second, the correct pBV or pJV clones were both digested withPac I and Asc I, and the resulting V_(H)-CH and V_(L)—CL-containing DNAfragments were ligated to form a single pBJ plasmid that contained bothheavy and light chain genes. The pBJ clones were screened by Srf I/Not Idigestion to confirm the presence of both antibody genes. The plasmidmap for Chagas 12-392-150 Mu-Hu_pBJ clone 4 is shown in FIG. 2; thedouble-stranded polynucleotide sequences of VH gene and VL genecontaining regions (and flanking sequences) are shown in FIGS. 3A-C.

CHO cell line B3.2 acquired from the Abbott Bioresearch Centercontaining a deficient dihydrofolate reductase (DHFR) gene was used fortransfection and stable antibody expression. CHO B3.2 cells weretransfected with Chagas 12-392-150 Mu-Hu_pBJ clone 1 using calciumphosphate-mediated transfection. The transfected CHO cells were culturedfor several weeks with media lacking thymidine to select for those cellsthat had incorporated the functional DHFR gene present in the pBJplasmid. Fluorescence-activated cell sorting (FACS) was used to sortindividual cells from the transfected pool into 96-well plates. Anantigen-specific EIA was used to rank antibody production among theclones, and the highest producers were expanded and re-assayed. Cloneswere then weaned into serum-free media. The growth characteristics,antibody production and clonality of the clones were monitored. ChagasFP3 clone 12-392-150 CHO 2580-104 was sub-cloned by sorting individualcells into 96-well plates and then expanded to produce purifiedantibody.

Example 6 Cell Lines Producing Chimeric Anti-T. cruzi Pep2 Epitope(Anti-TcF and Anti-FP6) mAbs (Chagas Pep2 Clone 9-638-1928)

Identification of Mouse V_(H) and V_(L) Sequences

Hybridoma cell line HBPep2 (Example 2) was cultured in H-SFM to obtain˜5×10⁶ cells for mRNA purification according to standard mRNA extractionprotocols. The purified mRNA was used as a template with a mouse Igprimer set (Novagen (EMD Biosciences, Inc.)) for a RT-PCR reaction.Positive PCR products were observed from the heavy chain (H) primers Band E (HB and HE clones) and from the light chain (L) primers B, C, D,E, F and G (LB, LC, LD, LE, LF and LG clones). All positive PCR productswere gel-purified and cloned into pCR TOPO 2.1 TA vector (InvitrogenCorp., Carlsbad, Calif.). The plasmid DNA was purified from transformedbacterial cells and the V_(H) or V_(L) inserts were confirmed by EcoRIdigestion for each RT-PCR reaction that generated appropriately sizedproducts. The correct V_(H) or V_(L) gene sequence was selected aftersequence alignments confirmed a consensus sequence among the clones.Chagas TOPO-TA clone HE2 contained the correct V_(H) gene sequence, andChagas TOPO-TA clone LG1 contained the correct V_(L) gene sequence.

Cloning Murine V_(H) and V_(L) Genes into pBOS Vectors

A pair of PCR primers containing a partial Kappa signal sequence and anNru I site on the 5′-primer, and a BsiW I site on the 3′-primer was usedto amplify the mouse V_(L) gene from TOPO clone LG1. Additionally, apair of primers containing a partial heavy chain signal sequence and anNru I site on the 5′-primer, and Sal I site on 3′-primer was used toamplify the mouse V_(H) gene from TOPO clone HE2. The V_(L) PCR productwas digested with Nru I and BsiW I restriction enzymes and ligated intopBOS-hCk vector digested with the same enzymes. The V_(H) PCR productwas digested with Nru I and Sal I restriction enzymes and ligated intopBOS-hCglvector digested with the same enzymes. Plasmids from a numberof transformed bacterial colonies were sequenced to confirm the presenceof either the Chagas V_(H) or V_(L) gene in their respective vectors.Chagas 9-638 V_(H) _(_)pBOS-H clone A2 and Chagas 9-638 V_(L) _(_)pBOS-Lclone B6 were deemed correct.

Chimeric mAb Production and Functional Confirmation

Endotoxin-free plasmid preparations of Chagas 9-638 V_(H) _(_)pBOS-Hclone A2 and Chagas 9-638 V_(L) _(_)pBOS-L clone B6 were used fortransient transfection into COS 7L cells by electroporation (GENEPULSER®, Bio-Rad, Hercules, Calif.). The transfected cells wereincubated at 37° C. in a 5% CO₂ incubator for three days. The chimericantibody produced by the COS 7L cells were harvested by centrifugationat 4000 rpm for 20 minutes and then purified using a protein A affinitycolumn (POROS A; Applied Biosystems). To confirm activity, the harvestedantibody was assayed using surface plasmon resonance on a BIACORE®instrument (Biacore (GE Healthcare); Piscataway, N.J.). Affinity wasapproximately 2.6 nM.

CHO Cell Line Stable Expression Vector Cloning

Chagas 9-638 V_(H) _(_)pBOS-H clone A2 and Chagas 9-638 V_(L) _(_)pBOS-Lclone B6 were used to construct a plasmid to generate a stable,transfected CHO cell line. First, Srf I and Not I were used to isolatethe V_(H)-CH and V_(L)-CL genes from the pBOS vectors; these fragmentswere then cloned into pBV or ply vectors, respectively. The resultingpBV and pJV clones were analyzed by Srf I/Not I restriction enzymedigestion and sequenced to determine Chagas 9-638 V_(H) _(_)pBV clone 10and Chagas 9-638_pJV clone 10 were correct. Second, the correct pBV orpJV clones were both digested with Pac I and Asc I, and the resultingV_(H)-CH and V_(L)-CL-containing DNA fragments were ligated to form asingle pBJ plasmid that contains both heavy and light chain genes. ThepBJ clones were screened by Srf I/Not I digestion to confirm thepresence of both antibody genes. The plasmid map for Chagas 9-638Mu-Hu_pBJ clone 2 is shown in FIG. 4.

CHO cell line B3.2 acquired from the Abbott Bioresearch Centercontaining a deficient DHFR gene was used for transfection and stableantibody expression. CHO B3.2 cells were transfected with Chagas 9-638Mu-Hu_pBJ clone 2 using calcium phosphate-mediated transfection. Thetransfected CHO cells were cultured for several weeks with media lackingthymidine to select for those cells that had incorporated the functionalDHFR gene present in the pBJ plasmid. FACS was used to sort individualcells from the transfected pool into 96-well plates. An antigen-specificEIA was used to rank antibody production among the clones, and thehighest producers were expanded and re-assayed. Clones were then weanedinto serum-free media. The growth characteristics, antibody productionand clonality of the clones were monitored. Chagas Pep2 clone 9-638-1145was chosen and re-subcloned by sorting individual cells into 96-wellplates, and then Chagas Pep2 clone 9-638-1928 expanded to producepurified antibody.

Example 7 Cell Lines Producing Chimeric Anti-T. cruzi FP10 mAbs (ChagasFP10 10-745-3796)

Identification of Mouse V_(H) and V_(L) Sequences

Hybridoma cell line HBFP10 (Example 3) was cultured in H-SFM to obtain˜5×10⁶ cells for mRNA purification according to standard mRNA extractionprotocols. The purified mRNA was used as a template with a mouse Igprimer set (Novagen (EMD Biosciences, Inc.)) for a RT-PCR reaction.Positive PCR products were observed from the heavy chain (H) primers B(HB clones) and from the light chain (L) primers B, C, and G (LB, LC andLG clones). All positive PCR products were gel-purified and cloned intopCR TOPO 2.1 TA vector (Invitrogen Corp., Carlsbad, Calif.). The plasmidDNA was purified from transformed bacterial cells and the V_(H) or V_(L)inserts were confirmed by EcoRI digestion for each RT-PCR reaction thatgenerated appropriately sized products. The correct V_(H) or V_(L) genesequence was selected after sequence alignments confirmed a consensussequence among the clones. Chagas TOPO-TA clone HB3 contained thecorrect V_(H) gene sequence, and Chagas TOPO-TA clone LG1 contained thecorrect V_(L) gene sequence.

Cloning Murine V_(H) and V_(L) Genes into pBOS Vectors

A pair of PCR primers containing a partial Kappa signal sequence and anNru I site on the 5′-primer, and a BsiW I site on the 3′-primer was usedto amplify the mouse V_(L) gene from TOPO clone LG1. Additionally, apair of primers containing a partial heavy chain signal sequence and anNru I site on the 5′-primer, and Sal I site on 3′-primer was used toamplify the mouse V_(H) gene from TOPO clone HB3. The V_(L) PCR productwas digested with Nru I and BsiW I restriction enzymes and ligated intopBOS-hCk vector digested with the same enzymes. The V_(H) PCR productwas digested with Nru I and Sal I restriction enzymes and ligated intopBOS-hCglvector digested with the same enzymes. Plasmids from a numberof transformed bacterial colonies were sequenced to confirm the presenceof either the Chagas V_(H) or V_(L) gene in their respective vectors.Chagas 10-745 V_(H) _(_)pBOS-H clone 4 and Chagas 10-745 V_(L)_(_)pBOS-L clone 5 were deemed correct.

Chimeric mAb Production and Functional Confirmation

Endotoxin-free plasmid preparations of Chagas 10-745 V_(H) _(_)pBOS-Hclone 4 and Chagas 10-745 V_(L) _(_)pBOS-L clone 5 were used fortransient transfection into COS 7L cells by electroporation (GENEPULSER®, Bio-Rad). The transfected cells were incubated at 37° C. in a5% CO₂ incubator for three days. The chimeric antibody produced by theCOS 7L cells were harvested by centrifugation at 4000 rpm for 20 minutesand then purified using a protein A affinity column (POROS A; AppliedBiosystems). To confirm activity, the harvested antibody was assayedusing surface plasmon resonance on a BIACORE® instrument (Biacore (GEHealthcare)).

CHO Cell Line Stable Expression Vector Cloning

Chagas 10-745 V_(H) _(_)pBOS-H clone 4 and Chagas 10-745 V_(L)_(_)pBOS-L clone 5 were used to construct a plasmid to generate astable, transfected CHO cell line. First, Srf I and Not I were used toisolate the V_(H)-CH and V_(L)-CL genes from the pBOS vectors; thesefragments were then cloned into pBV or ply vectors, respectively. Theresulting pBV and pJV clones were analyzed by Srf I/Not I restrictionenzyme digestion and sequenced to determine Chagas 10-745 V_(H) _(_)pBVclone 1 and Chagas 10-745_pJV clone 1 were correct. Second, the correctpBV or pJV clones were both digested with Pac I and Asc I, and theresulting V_(H)-CH and V_(L)-CL-containing DNA fragments were ligated toform a single pBJ plasmid that contains both heavy and light chaingenes. The pBJ clones were screened by Srf I/Not I digestion to confirmthe presence of both antibody genes. The plasmid map for Chagas 10-745Mu-Hu_pBJ clone 1 is shown in FIG. 5.

CHO cell line B3.2 acquired from the Abbott Bioresearch Centercontaining a deficient DHFR gene was used for transfection and stableantibody expression. CHO B3.2 cells were transfected with Chagas 10-745Mu-Hu_pBJ clone 1 using calcium phosphate-mediated transfection. Thetransfected CHO cells were cultured for several weeks with media lackingthymidine to select for those cells that had incorporated the functionalDHFR gene present in the pBJ plasmid. FACS was used to sort individualcells from the transfected pool into 96-well plates. An antigen-specificEIA was used to rank antibody production among the clones, and thehighest producers were expanded and re-assayed. Clones were then weanedinto serum-free media. The growth characteristics, antibody productionand clonality of the clones were monitored. Chagas FP10 clone10-745-3649 was sub-cloned by sorting individual cells into 96-wellplates and then expanded to produce purified antibody.

Example 8 Cell Lines Producing Chimeric Anti-T. cruzi FRA mAbs(Prophetic Example)

Identification of Mouse V_(H) and V_(L) Sequences

Hybridoma cell line HBFRA (Example 4) is cultured in H-SFM to obtain˜5×10⁶ cells for mRNA purification according to standard mRNA extractionprotocols. The purified mRNA is used as a template with a mouse Igprimer set (Novagen (EMD Biosciences, Inc.)) for a RT-PCR reaction.Positive PCR products are observed from the heavy chain (H) primers andfrom the light chain (L) primers. All positive PCR products aregel-purified and cloned into pCR TOPO 2.1 TA vector (Invitrogen Corp.,Carlsbad, Calif.). The plasmid DNA is purified from transformedbacterial cells and the V_(H) or V_(L) inserts are confirmed by EcoRIdigestion for each RT-PCR reaction that generated appropriately sizedproducts. The correct V_(H) or V_(L) gene sequence is selected aftersequence alignments confirm a consensus sequence among the clones.

Cloning Murine V_(H) and V_(L) Genes into pBOS Vectors

A pair of PCR primers containing a partial Kappa signal sequence and anNru I site on the 5′-primer, and a BsiW I site on the 3′-primer is usedto amplify the mouse V_(L) gene from TOPO. Additionally, a pair ofprimers containing a partial heavy chain signal sequence and an Nru Isite on the 5′-primer, and Sal I site on 3′-primer is used to amplifythe mouse V_(H) gene from TOPO clone. The V_(L) PCR product is digestedwith Nru I and BsiW I restriction enzymes and ligated into pBOS-hCkvector digested with the same enzymes. The V_(H) PCR product is digestedwith Nru I and Sal I restriction enzymes and ligated intopBOS-hCglvector digested with the same enzymes. Plasmids from a numberof transformed bacterial colonies are sequenced to confirm the presenceof either the Chagas V_(H) or V_(L) gene in their respective vectors(Chagas V_(H) _(_)pBOS-H and Chagas V_(L) _(_)pBOS-L).

Chimeric mAb Production and Functional Confirmation

Endotoxin-free plasmid preparations of Chagas V_(H) _(_)pBOS-H andChagas V_(L) _(_)pBOS-L are used for transient transfection into COS 7Lcells by electroporation (GENE PULSER®, Bio-Rad) or other transfectionmethod. The transfected cells are incubated at 37° C. in a 5% CO₂incubator for about three days. The chimeric antibody produced by theCOS 7L cells is harvested by centrifugation at 4000 rpm for 20 minutesand then purified using a protein A affinity column (POROS A; AppliedBiosystems; Foster City, Calif.). To confirm activity, the harvestedantibody is assayed by, for example using surface plasmon resonance on aBIACORE® instrument (Biacore (GE Healthcare)).

CHO Cell Line Stable Expression Vector Cloning

Chagas V_(H) _(_)pBOS-H and Chagas V_(L) _(_)pBOS-L are used toconstruct a plasmid to generate a stable, transfected CHO cell line.First, Srf I and Not I are used to isolate the V_(H)-CH and V_(L)-CLgenes from the pBOS vectors; these fragments are then cloned into pBV orpJV vectors, respectively. The resulting pBV and pJV clones are analyzedby Srf I/Not I restriction enzyme digestion and sequenced to determinethat the clones are correct. Second, the correct pBV or pJV clones areboth digested with Pac I and Asc I, and the resulting V_(H)-CH andV_(L)-CL-containing DNA fragments are ligated to form a single pBJplasmid that contains both heavy and light chain genes. The pBJ clonesare screened by Srf I/Not I digestion to confirm the presence of bothantibody genes, resulting in Chagas Mu-Hu_pBJ.

A CHO cell line, such as CHO B3.2, containing a deficient DHFR gene isused for transfection and stable antibody expression. CHO B3.2 cells aretransfected with Chagas Mu-Hu_pBJ using calcium phosphate-mediatedtransfection or other transfection protocol. The transfected CHO cellsare cultured for several weeks with media lacking thymidine to selectfor those cells that incorporate the functional DHFR gene present in thepBJ plasmid. FACS can be used to sort individual cells from thetransfected pool into 96-well plates. An antigen-specific EIA can beused to rank antibody production among the clones, and the highestproducers are expanded and re-assayed. Clones are then weaned intoserum-free media. The growth characteristics, antibody production andclonality of the clones are monitored. If desired, cell line clones canbe sub-cloned by sorting individual cells into 96-well plates and thenexpanded to produce purified antibody.

Example 9 Kinetics/Affinity Determination of Recombinant Chimeric ChagasAntibody for Chagas Antigen TcF

The kinetics/affinity were determined using a high density, goatanti-human IgG Fc capture biosensor on a BIAcore 2000. The flow cellswere first equilibrated with a running buffer composed of HBS-EP spikedwith 6 g/L of Carboxymethyl-Dextran (hereinafter referred to as a“Running Buffer”) (Fluka) and 6 g/L BSA for 5 minutes at flow rate of 10μL/minutes. Next, recombinant chimeric anti-Chagas monoclonal antibody,namely, 9-638-132 (Pep2 epitope in TcF and FP6), 10-745-140 (FP10) and12-392-150 (FP3), each diluted into Running Buffer, were injected overindividual flow cells and captured by the biosensor with one flow cellleft blank as a reference flow cell. The buffer flow rate was increasedto 100 μL/minute and the flow cells were washed for 10 minutes prior toa 150 μL injection of the antigen at various concentrations from 0 to100 nM in Running Buffer followed by Running Buffer alone for 60 to 360seconds. The anti-human IgG capture biosensor was then regenerated withthree 33 μL injections of 100 mM H₃PO₄ and the steps above were repeateduntil all concentrations of each Chagas antigen were tested induplicate. The binding kinetics, association (k_(a)) and dissociation(k_(d)), were monitored for each antigen injection by sensorgrams andthe kinetics/affinity were determined by Scrubber 2.0 software (BioLogicSoftware Pty Ltd., Australia). The interactions between the recombinantchimeric anti-Chagas monoclonal antibodies with the Pep2 epitope withinthe Chagas TcF antigen are shown below in Table 14.

TABLE 14 Chimeric Chagas Ab k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) 9-638-132 4.0 × 10⁶ 1.7 × 10⁻² 4.1 × 10⁻⁹ 10-745-140 No binding wasobserved. 12-392-150

Example 10 Kinetics/Affinity Determination of Recombinant ChimericChagas Antibody for Chagas Antigens FP3 and FP10

The kinetics/affinity were determined using a high density, anti-His₄capture biosensor on a BIAcore 2000. The flow cells were firstequilibrated with a Running Buffer (as defined above in Example 9)composed of HBS-EP buffer spiked with 1% BSA and 1% Tween 20 for 5minutes at a flow rate 50 μL/minute. Next, Chagas antigens (each antigencontains a His₆ tag), namely FP10 and FP3, were each diluted intoRunning Buffer, injected over individual flow cells, and captured by thebiosensor with one flow cell left blank as a reference flow cell. Thebuffer flow rate was increased to 100 μL/minute and the flow cells werewashed for 5 minutes prior to a 150 μL injection of each of therecombinant Chimeric anti-Chagas monoclonal antibodies, namely,9-638-132 (Pep2 epitope in TcF and FP6), 10-745-140 (FP10) and12-392-150 (FP3), at various concentrations from 0 to 300 nM in RunningBuffer followed by Running Buffer alone for 60 to 360 seconds. Theanti-His₄ capture biosensor was then regenerated with two 35 μLinjections of Gentle Ab/Ag Elution Buffer (Pierce) spiked with 2.5 mMH₃PO₄ and two 25 μL injections of 5 mM H₃PO₄ and the steps above wererepeated until all concentrations of each Chimeric anti-Chagas antibodywere tested in duplicate. The binding kinetics, association (k_(a)) anddissociation (k_(d)) were monitored for each antibody injection bysensorgrams and the kinetics/affinity were determined by Scrubber 2.0software (BioLogic Software Pty Ltd., Australia). The interactionsbetween the recombinant chimeric anti-Chagas monoclonal antibodies withthe Chagas FP10 antigen are shown below in Table 15. The interactionsbetween the recombinant chimeric anti-Chagas monoclonal antibodies withthe Chagas FP3 antigen are shown below in Table 16.

TABLE 15 Chimeric Chagas Ab k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) 9-638-132 No binding was observed. 10-745-140 1.2 × 10⁵ 3.6 × 10⁻⁴ 2.9× 10⁻⁹ 12-392-150 No binding was observed.

TABLE 16 Chimeric Chagas Ab k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) 9-638-132 No binding was observed. 10-745-140 12-392-150 2.7 × 10⁶ 3.8× 10⁻⁴ 1.4 × 10⁻¹⁰

Example 11 Kinetics/Affinity Determination of Murine Chagas Antibody forChagas Antigens

The kinetics/affinity were determined using a high density, rabbitanti-mouse IgG capture biosensor on a BIAcore 2000. The flow cells werefirst equilibrated with a Running Buffer composed of HBS-EP bufferspiked with 1% BSA, 1% Carboxymethyl-Dextran (“Running Buffer”) (Fluka),and 0.1% Tween 20 at 5 μL/minute for 5 minutes. Next, each murineanti-Chagas antibody (namely, monoclonal antibodies (mAbs) 8-367-171(FRA), 9-638-132 (Pep2 epitope in TcF and FP6), 10-745-140 (FP10) and12-392-150 (FP3) diluted into Running Buffer, was injected overindividual flow cells and captured by the biosensor. The buffer flowrate was increased to 60 al/min and the flow cells were washed for 5minutes prior to a 150 μL injection of Chagas antigen at variousconcentrations from 0 to 200 nM in Running Buffer followed by RunningBuffer alone for 60 to 360 seconds. The flow rate was then changed to 10μL/minute and the anti-mouse IgG capture biosensor was then regeneratedwith one 30 μL injection of 10 mM Glycine pH 1.7 and the steps abovewere repeated until all concentrations of each Chagas antigen weretested in duplicate. The binding kinetics, association (k_(a)) anddissociation (k_(d)) were monitored for each antigen injection bysensorgrams and the kinetics/affinity were determined by Scrubber 2.0software (BioLogic Software Pty Ltd., Australia).

For Chagas antigens FRA, FP6, TcF, and FP3, the flow cell containinganti-Chagas mAb 10-745-140 was used as the reference flow cell. The flowcell containing anti-Chagas mAb 9-638-132 was used as the reference flowcell for Chagas antigen FP10. The interaction between the monoclonalanti-Chagas antibodies with the Chagas FRA antigen itself is shown belowin Table 17. The interaction between the monoclonal anti-Chagasantibodies with the FRA and the Chagas PEP2 epitope of the Chagas FP6antigen is shown below in Table 18. The interaction between themonoclonal anti-Chagas antibodies with the Chagas PEP2 epitope of theChagas TcF antigen is shown below in Table 19. The interaction betweenthe monoclonal anti-Chagas antibodies with the Chagas FP10 antigen isshown below in Table 20. The interaction between the monoclonalanti-Chagas antibodies with the Chagas FP3 antigen is shown below inTable 21.

TABLE 17 Murine Chagas Ab k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) 8-367-171 3.6 × 10⁶ 1.3 × 10⁻¹ 3.7 × 10⁻⁸  9-638-132 No binding wasobserved 10-745-140 12-392-150

TABLE 18 Murine Chagas Ab k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) 8-367-171 1.5 × 10⁶ 7.9 × 10⁻³ 5.2 × 10⁻⁹  9-638-132 Binding wasobserved, but could not be fit to 1:1 model 10-745-140 No binding wasobserved 12-392-150

TABLE 19 Murine Chagas Ab k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) 8-367-171 No binding was observed  9-638-132 2.1 × 10⁶ 1.2 × 10⁻² 5.7 ×10⁻⁹ 10-745-140 No binding was observed 12-392-150

TABLE 20 Murine Chagas Ab k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) 8-367-171 No binding was observed  9-638-132 10-745-140 1.1 × 10⁵ 2.2 ×10⁻⁴ 1.9 × 10⁻⁹ 12-392-150 No binding was observed

TABLE 21 Murine Chagas Ab k_(a) (M⁻¹s⁻¹) k_(d) (s⁻¹) K_(D) (M) 8-367-171 No binding was observed  9-638-132 10-745-140 12-392-150 5.6× 10⁵ 5 × 10⁻⁵ 8 × 10⁻¹¹

One skilled in the art would readily appreciate that the presentdisclosure is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. Themolecular complexes and the methods, procedures, treatments, molecules,specific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the disclosure. It will be readily apparentto one skilled in the art that varying substitutions and modificationsmay be made to the disclosure disclosed herein without departing fromthe scope and spirit of the disclosure.

All patents and publications mentioned in the specification areindicative of the levels of those skilled in the art to which thedisclosure pertains. All patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

What is claimed is:
 1. An immunodiagnostic reagent comprising a monoclonal antibody that specifically binds to a diagnostically relevant region of a T cruzi FP10 polypeptide, or an antigen binding fragment thereof, which monoclonal antibody comprises a variable light chain region (V_(L)) amino acid sequence of SEQ ID NO: 18 and a variable heavy chain region (V_(H)) amino acid sequence of SEQ ID NO:
 20. 2. A monoclonal antibody that specifically binds to diagnostically relevant region of a T cruzi FP10 polypeptide, or an antigen-binding fragment thereof, which monoclonal antibody comprises a variable light chain region (V_(L)) amino acid sequence of SEQ ID NO: 18 and a variable heavy chain region (V_(H)) amino acid sequence of SEQ ID NO:
 20. 3. A cell line that expresses a chimeric antibody that specifically binds to a diagnostically relevant region of a T cruzi FP10 polypeptide, wherein said cell line is deposited with the American Type Tissue Collection and identified by patent deposit designation selected PTA-8140.
 4. A method of purifying a T cruzi FP10 polypeptide comprising the amino acid sequence of SEQ ID NO: 6, which method comprises: (a) contacting a sample suspected of containing a T cruzi FP10 polypeptide with the immunodiagnostic reagent according to claim 1 under conditions that allow formation of antibody:antigen complexes; (b) isolating the antibody:antigen complexes formed; and (c) separating the antigen from the antibody.
 5. The immunodiagnostic reagent of claim 1, which comprises an antigen-binding fragment of the monoclonal antibody selected from a Fab fragment, a Fab′ fragment, a Fab′-SH fragment, a F(ab′)2 fragment, an Fv fragment, a diabody, and a single-chain Fv (scFv) molecule.
 6. The monoclonal antibody of claim 2, which comprises an antigen-binding fragment of the monoclonal antibody selected from a Fab fragment, a Fab′ fragment, a Fab′-SH fragment, a F(ab′)2 fragment, an Fv fragment, a diabody, and a single-chain Fv (scFv) molecule. 